NF-κB Is Transported into the Nucleus by Importin α3 and Importin α4*

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-B 1 p50/p65 transcription factor has a central role in controlling host cell immune and inflammatory responses, cell differentiation, and apoptosis (1,2). Dysregulation of NF-B has been associated with several common diseases such as cancer and diabetes (3). Cytoplasmic NF-B 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-B 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-B activation (2,4).
NF-B transcription factors are dimers belonging to the Rel family (5). All five mammalian NF-B 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 (6,7). NF-B subunits can form various dimers, but the classical, best characterized form is composed of p50 and p65 (1,7,8). 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 (9).
NF-B dimer is held in an inactive state in the cytoplasm by an inhibitor protein (IB) that masks the NLSs of the subunits (10 -13). IB␣ preferentially inhibits the nuclear translocation of the p50/p65 heterodimer. Other IB molecules found in higher vertebrates include IB␤, IB⑀, and Bcl3. All IB molecules contain ankyrin repeats, which mediate specific interactions with the Rel-homology domains of NF-B molecules. The C-terminal regions of p100 and p105 proteins also contain ankyrin repeats and they can function as an IB (2,4,14). Crystal structures of most NF-B Rel domains bound to DNA or IB have been determined (15)(16)(17)(18)(19)(20)(21)(22).
p50/p65 heterodimers and p50 homodimers are considered the most abundant NF-B 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 IB (IB␥). Additional post-translational steps regulate p50 homodimer formation (14,29,31). Under nonstimulated conditions 10 -20% of p105 proteins are processed to form p50 homodimers (32). 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 (18,31).
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 (Ͼ25 nm in diameter) is regulated by specific nuclear import and export systems. Proteins that contain classical NLSs are imported into the nucleus by importin ␣/␤ heterodimers. Importin ␣ binds to NLS containing proteins, and importin ␤ 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 (33,34). A classical monopartite NLS consists of a stretch of basic amino acids, arginines and lysines (35,36). Classical NLSs are found in p50 and p65 (37,38). 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 (39).
We now report that TNF-␣-induced nuclear import of NF-B p50/p65 heterodimers is mediated by importin ␣3 and importin ␣4. Importin ␣3 is also involved in uninduced import of p50 homodimers. Importin ␣ 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 ␣3.
Cells, Media, and Other Reagents-Human A549 lung carcinoma cell line (ATCC, CCL 185) was maintained in continuous culture in minimum Eagle's medium-␣ (Invitrogen) supplemented with 0.6 g/ml penicillin, 60 g/ml streptomycin, and 10% fetal calf serum (Integro, Zaandam, the Netherlands). Human hepatocellular carcinoma HuH7 (49) cells were maintained in minimum Eagle's medium-␣ 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-␣ (TNF-␣) was purchased from R&D systems (Abingdon, UK). For cell stimulation 5 ng/ml of TNF-␣ was used. Leptomycin B (LMB) (10 g/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 (50). For in vitro translation 35 S-labeled PRO.MIX (Ͼ100 Ci/mmol) was used, and it was obtained from Amersham Biosciences (Buchinghamshire, UK).
Plasmids and DNA Manipulations-Escherichia coli-produced GSTimportins ␣1, ␣3, ␣5, ␣7, and ␤ as well as the mutants in the arm repeats 3 and 8 of importin ␣3 have been described previously (48). To create arm repeat 7 mutations to GST-importin ␣3 we used Quick-Change TM site-directed mutagenesis kit (Stratagene). The primer used was 5Ј-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 5Ј-CAA AGA TAA AGA AGA AGT GCA GAG GGC AGC TCA GAA GCT CAT GCC CAA TTT TTC G (p50 NLS: K362A, R363A) and 5Ј-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(ϩ) (51) and pcDNA3.1/Myc-His(Ϫ) (Invitrogen) expression vectors. The primers used were 5Ј-ATA TAT AGA TCT ACC ATG GCA GAA GAT GAT CCA TAT TTG (5Ј-oligonucleotide, BglII codons in bold face and initiation codon underlined) and 5Ј-ATA TAT AGA TCT TTA CAT GGT TCC ATG CTT CAT CCC AGC ATT AGA TTT AG (3Ј-oligonucleotide) for p50 and 5Ј-ATA TAT GGA TCC ACC ATG GAC GAA CTG TTC CCC CTC ATC (5Ј-oligonucleotide, BamHI codons in bold face) and 5Ј-ATA TAT GGA TCC GGA GCT GAT CTG ACT CAG CAG GGC (3Ј-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 5Ј-A GAA AAC (AGA TCT) ACC ATG TCC CCT ATA CTA GGT TAT TG (5Ј-oligonucleotide) and 5Ј-G TGG TGC (AGA TCT) TTA GGA TCC ACG CGG AAC CAG ATC CGA TTT TG (3Ј-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 (50).
For protein production of GST-tagged human importin ␣1, ␣3, ␣4, ␣7, 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-␣ (5 ng/ml) or left nonstimulated. The cells were washed, harvested, and lysed in L-buffer containing 1 mM NaVO 4 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 ϩ4°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 l of glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences) at ϩ4°C for 60 min in L-buffer followed by washing twice with the buffer. 25 l of glutathione-Sepharose-immobilized GST fusion proteins was mixed with 200 l of cell lysate and rotated at ϩ4°C for 2 h followed by washing three times with L-buffer. Sepharose beads were dissolved in 30 l of 2ϫ Laemmli sample buffer, and the proteins were separated on 10% SDS-PAGE (52). 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 l of in vitro translated p50, p65, or IB␣ proteins (TNT-coupled reticulocyte lysate systems, Promega) were allowed to bind to 25 l of Sepharose-immobilized GST-importin on ice for 60 min followed by washing three times with L-buffer. GST importin-bound 35 S-labeled proteins were dissolved in 30 l of 2ϫ 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 l of protein A-Sepharose (Amersham Biosciences) was incubated with 5 g of goat immunoglobulins against NF-B p50 or NF-B 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-␣ (5 ng/ml) for 30 min or left nonstimulated), rotated at ϩ4°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 ␣3, and IB␣.
Oligonucleotide Precipitation-A549 cells were stimulated with 5 ng/ml of TNF-␣ for 0, 15, 30, or 60 min. The cells were collected, and samples were treated as described by Rosen et al. (53). Upper strands of CCL5 (RANTES) (5Ј-gga tcc CTC CCC TTA GGG GAT GCC CCT CAA CT) and CXCL10 (IP-10) (5Ј-gga tcc GCA GAG GGA AAT TCC GTA ACT TGG) promoter NF-B elements were synthesized with BamHI overhangs as spacers, and they were 5Ј-biotinylated (DNA Technologies Inc., Gaithersburg, MD). Lower strands were nonbiotinylated. Oligonucleotides were annealed in 0.5 M NaCl and incubated with streptavidinagarose beads (Neutravidin; Pierce) at ϩ4°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 ϩ4°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 NaVO 4 , and protease inhibitors. After washing oligonucleotide-bound proteins were dissolved in 75 l of 2ϫ 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) (54) for 20 min followed by stimulation with TNF-␣ (100 ng/ml) in the presence of LMB (2 ng/ml) for 30 min. The cells were fixed with methanol at Ϫ20°C for 10 min and processed for immunofluorescence as previously described (55). 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.

RESULTS
NF-B Binds to Importin ␣3 and Importin ␣4 -Upon activation NF-B is translocated from the cytoplasm into the nucleus. The nucleocytoplasmic shuttling of NF-B has been thought to be mediated via importin ␣/␤and exportin 1-dependent pathways. However, the molecular mechanisms of the nuclear import of NF-B have remained unknown. To investigate the possible interactions of NF-B with different importin isoforms, we stimulated human A549 lung carcinoma cells with TNF-␣. Different expression systems were used for importin ␣3, 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 ␣1, ␣3, ␣5, ␣7, or ␤ (Fig. 1A) or baculovirus-expressed GST-importins ␣1, ␣3, ␣4, or ␣7 (Fig. 1B). Proteins bound to GST-importins were analyzed by Western blotting for the presence of p50 and p65. As shown in Fig. 1A, p50 and p65 proteins bound strongly to bacterially expressed importin ␣3 and to a lesser extent to importin ␣1, whereas no binding to importins ␣5, ␣7, or ␤ was detected. Small amounts of p50 bound to importin ␣3 also under nonstimulated conditions. When we used baculovirus-expressed importins in pull-down experiments we found that p50 and p65 bound to importin ␣3 and ␣4 whereas no binding to importin ␣1 or ␣7 was detected (Fig. 1B). Bacterially expressed importin ␣3 was used in all further experiments.
To characterize the binding of intrinsic cellular importins to NF-B proteins we used baculovirus-expressed GST-p50 and GST-p65 in pull-down experiments. Cell extracts were prepared from cultured A549 cells and the proteins in the cell FIG. 1. TNF-␣-activated NF-B binds to importin ␣3 and ␣4. A and B, cultured A549 cells were stimulated with 5 ng/ml of TNF-␣ for 30 min or left untreated as indicated. Cell extracts were prepared, and the proteins in the cell extracts were allowed to bind to Sepharose-immobilized bacterially expressed GST-importins ␣1, ␣3, ␣5, ␣7, or ␤ (A) or baculovirus-expressed GST-importins ␣1, ␣3, ␣4, or ␣7 (B) at ϩ4°C for 2 h. Sepharose-bound proteins were dissolved in Laemmli sample buffer followed by SDS-PAGE and Western blotting with anti-p50 or anti-p65 antibodies. A similar gel was also stained with Coomassie Blue to visualize the amount of Sepharose-immobilized GST-importin isoforms. C, to determine the specificity of anti-importin antibodies bacterially expressed GST-importins ␣1, ␣3, ␣5, and ␣7 were separated on SDS-PAGE followed by Western blotting with anti-importin ␣1 and ␣3 antibodies. D, cell extracts were prepared from cultured A549 cells, and the proteins in the cell extracts were allowed to bind to Sepharoseimmobilized GST-p50 and GST-p65 proteins (first lane shows uninfected Sf9 cell extract bound to Sepharose as a control). Sepharosebound proteins were dissolved in Laemmli sample buffer followed by SDS-PAGE and Western blotting with anti-importin ␣1 and ␣3 antibodies.
extracts were allowed to bind to Sepharose-immobilized GST-p50 and GST-p65 proteins followed by Western blotting for the presence of importin ␣1 and ␣3. As is shown in Fig. 1D, importin ␣3 bound to Sepharose-immobilized GST-p50 and GST-p65 proteins. We did not observe any importin ␣1 binding to GST-p50 or GST-p65.
We wanted to verify these in vitro results with in vivo experiments made by immunoprecipitation. Cultured A549 cells were stimulated with TNF-␣ or left nonstimulated. Cell extracts were prepared and the proteins in the cell extracts were allowed to bind to Protein A Sepharose-immobilized anti-p50 or anti-p65 immunoglobulins. Bound proteins were analyzed by Western blotting for the presence of p50, p65, importin ␣3, and IB␣. As is shown in Fig. 2, importin ␣3 coprecipitated with p50 and p65 only after TNF-␣ stimulation when the complex was free of IB␣.
Kinetics of TNF-␣-induced p50/p65 Activation-Inactive p50/ p65 heterodimers preexists in the cytoplasm associated with their specific inhibitory molecule IB␣. A diverse range of stimuli, including TNF-␣ induction, results in serine phosphorylation of IB␣ and its subsequent degradation followed by translocation of the free p50/p65 heterodimer into the nucleus and activation of genes containing NF-B binding sites. It has been suggested that in nonstimulated cells NF-B⅐IB␣ complexes are shuttling between the nucleus and cytoplasm (56 -59). It has been proposed that this shuttling is mediated by the NLS of p50 that would be exposed in the NF-B⅐IB␣ complex (60). To investigate the kinetics of NF-B binding to importin ␣3, we stimulated A549 cells with TNF-␣ for different time periods (Fig. 3). Whole cell extracts were prepared, and the proteins in the cell extracts were analyzed by Western blotting with anti-p50, anti-p65, anti-IB␣ and anti-phospho(Ser 32 )-IB␣ (P-IB␣) antibodies. The protein levels of p50/p105 and p65 remained constant throughout the experiment (Fig. 3A). IB␣ was degraded rapidly after TNF-␣ induction. IB␣ levels started to decrease at 1 min after TNF-␣ stimulation, and the protein was completely degraded at 8 min after TNF-␣ stimulation. The appearance of the phosphorylated form of IB␣ was also a very rapid phenomenon. After 1 min of TNF-␣ stimulation the phosphorylation of IB␣ was detectable and it was strongly phosphorylated after 4 min of induction. The phospho-rylated form of IB␣ disappeared after 8 min of TNF-␣ stimulation due to the degradation of IB␣ (Fig. 3A). To further investigate the ability of NF-B to bind to importin ␣3 the proteins in the cell extracts were allowed to bind to Sepharoseimmobilized GST-importin ␣3. Unbound protein was washed away, and the bound proteins were analyzed by Western blotting for the presence of p50 and p65. As shown in Fig. 3B, p65 binding to importin ␣3 was seen only after IB␣ was degraded (Ն8 min) from the NF-B⅐IB␣ complexes.
Importin ␣3 bound small amounts of p50 also under nonstimulated conditions (Figs. 1A and 3B). The complex containing p50 under nonstimulated conditions did not include p105 (Fig. 3B). A similar gel as in Fig. 3B was also stained with anti-IB␣ antibodies but no IB␣ was detectable in the protein complexes bound to importin ␣3 (results not shown, Fig. 5). This suggests that the p50 protein bound to importin ␣3 under nonstimulated conditions is in the form of free p50 homodimer. No p65 binding to any importin ␣ isoform under nonstimulated conditions was seen (Figs. 1A and 3B).
To see whether p50 homodimers were also able to bind DNA under nonstimulated conditions we carried out oligonucleotide precipitation experiments using NF-B promoter elements. A549 cells were stimulated with TNF-␣ for 0, 15, 30, and 60 min. Cellular proteins were precipitated with oligonucleotides containing NF-B binding sites from CCL5 and CXCL10 genes followed by Western blotting with anti-p50 and anti-p65 antibodies. p65 containing dimers were bound to NF-B promoter elements only after TNF-␣ stimulation, but small amounts of p50 homodimers were also bound under nonstimulated conditions (Fig. 4). These data are well in line with the experiments described in Figs. 1A, 3B, 5A, and 7C.
IB␣ Inhibits the Binding of p65 Homodimers and p50/p65 Heterodimers but Not p50 Homodimers to Importin ␣3-To further characterize NF-B binding to importins ␣3 and ␣1, we used in vitro translated p50 and p65 proteins in GST-importin binding experiments. In vitro translated p50 homodimers are FIG. 2. Importin ␣3 is coprecipitated with p50 and p65. A549 cells were stimulated with 5 ng/ml of TNF-␣ for 30 min or left nonstimulated. Cell extracts were prepared and the proteins in the cell extracts were allowed to bind to protein A-Sepharose-immobilized anti-p50 (A) or anti-p65 (B) immunoglobulins. Bound proteins were analyzed by Western blotting for the presence of p50, p65, importin ␣3, and IB␣.

FIG. 3. Kinetics of TNF-␣-induced p50/p65 activation.
A549 cells were stimulated with 5 ng/ml of TNF-␣ for 1 to 120 min or left untreated as indicated. A, cell extracts were prepared, and the proteins in the cell extracts (15 g) were analyzed by Western blotting with specific antibodies. B, proteins in the cell extracts were allowed to bind to Sepharose-immobilized GST-importin ␣3 at ϩ4°C for 2 h. Sepharose-bound proteins were dissolved in Laemmli sample buffer, followed by SDS-PAGE and Western blotting with anti-p50 or anti-p65 antibodies. Coomassie Blue-stained gel is shown to visualize the amount of GSTimportin ␣3.
First, we wanted to ensure that in vitro translated proteins behave in a similar fashion to native TNF-␣-activated NF-B proteins in importin ␣ binding experiments (Fig. 1). We observed that also in vitro translated p50 homodimers bound strongly to importin ␣3 and to a lesser extent to importin ␣1 (Fig. 5A). p65 homodimers were bound strongly to importin ␣3 but no marked binding to importin ␣1 was seen (Fig. 5A).
Because it has previously been shown that IB␣ is also found in the nucleus, we wanted to study whether IB␣ uses the importin ␣/␤ import machinery and binds to importins. As is seen in Fig. 5A, no IB␣ binding to any GST-importins was detected, which support the previous findings that IB␣ is not transported into the nucleus by the classical importin ␣/␤ pathway. Nuclear import of IB␣ may be mediated through direct interactions with components of the NPC (61), or it is transported into the nucleus by a piggy-back mechanism with NLScontaining proteins (62).
Because it is well-known that IB␣ inhibits nuclear translocation of NF-B, we wanted to analyze whether IB␣ could inhibit the binding of NF-B in our importin ␣3 binding assay. IB␣ preferentially binds to p50/p65 heterodimers and with slightly lower affinity to p65 homodimers and with significantly lower affinity to p50 homodimers (63). As shown in Fig.  5B, IB␣ inhibited the binding of p65 homodimers but not p50 homodimers to importin ␣3. IB␣ also inhibited the binding of p50/p65 heterodimers to importin ␣3 (p50ϩp65 in Fig. 5B).
IB␣ was not found in a complex with the NF-B dimers that were bound to Sepharose-immobilized GST-importin ␣3 (Fig.  5B). This, together with the data in Figs. 2 and 3, imply that the NF-B complexes that are bound to importin ␣3 and are further transported into the nucleus are free of IB␣.
Classical NLSs of p50 and p65 Mediate Interactions with Importins ␣3 and ␣1-p50 and p65 proteins have been shown to contain arginine/lysine-rich NLSs (37,38). To study whether these elements are involved in direct binding of p50 and p65 proteins to importins ␣3 and ␣1, we carried out binding experiments with E. coli-produced GST-importins and in vitro translated p50 and p65 proteins. In vitro translated [ 35 S]methionine-labeled wild-type or NLS mutant p50 (K362A, R363A) and p65 (K301A, R302A, K303A) were allowed to bind to Sepharose-immobilized GST-importins ␣3 or ␣1 followed by identification of importin bound p50 and p65 by SDS-PAGE and autoradiography. Wild-type p50 and p65 proteins bound to importins ␣3 and ␣1 whereas NLS-mutated p50 and p65 completely failed to bind to either importin ␣ isoform (Fig. 6, A and  B). To confirm the biochemical observations also in cultured cells the nuclear import of the wild-type and NLS-mutated p50 and p65 was studied in transiently transfected HuH7 cells by indirect immunofluorescence microscopy. As seen in Fig. 6C, wild-type p50 and p65 were transported into the nucleus whereas NLS-mutated proteins were not. These data show that the NLSs of p50 and p65 mediate the binding of NF-B to importins ␣3 and ␣1 and its subsequent nuclear localization. p65 is known to exhibit a strong CRM1-dependent nuclear export signal (NES), which mediates its transport back to the cytoplasm. An inhibitor specific for CRM1-dependent export, LMB was used in Fig. 6C to inhibit the nuclear export of p65.
p50 Is Bound to the N terminus and p65 to the C-terminal NLS Binding Site of Importin ␣3-Importin ␣s are relatively well conserved molecules (Fig. 7A) that contain 10 arm repeats, which mediate the interactions with NLS-containing proteins. N-terminal arm repeats 2-4 have been considered as the major NLS binding site and C-terminal arm repeats 7-9 have been referred to as the minor NLS binding site (46,47) (Fig. 7B). An alignment of the arm repeats comprising the N-and C-terminal NLS binding sites shows that the tryptophan and asparagine residues are conserved in all arm repeats except arm 9. The primary sequence of the surrounding residues is not conserved between the arm repeats although the arm repeats are conserved in sequence and order when human importins are compared with each other (Fig. 7, A and B). To further characterize the mechanism of NF-B binding to importin ␣3 molecule, we created mutations in the N-terminal and C-terminal NLS binding sites of importin ␣3. Proteins in TNF-␣-stimulated or nonstimulated A549 cell extracts were allowed to bind to wild type or arm 3-or arm 7ϩ8-mutated importin ␣3 molecules followed by analysis of bound p50 and p65 by Western blotting. Mutations in the arm repeat 3 had no effect on p65 binding, whereas mutations in the arm repeats 7ϩ8 lead to an almost complete inhibition of p65 binding (Fig. 7C). Unlike in the case of p65, arm repeat 3 mutations in importin ␣3 completely prevented FIG. 4. Binding of TNF-␣-activated p50 and p65 proteins to CCL5 and CXCL10 promoter NF-B elements. A549 cells were stimulated with 5 ng/ml of TNF-␣ for 0, 15, 30, or 60 min as indicated. The cells were collected, and the proteins in the cell lysates were allowed to bind to biotin-labeled CCL5 (RANTES) or CXCL10 (IP-10) promoter NF-B oligonucleotides. The complexes were separated by streptavidin beads, and Sepharose-bound proteins were analyzed by Western blotting with anti-p50 and anti-p65 antibodies.
FIG. 5. IB␣ inhibits the binding of p65 homodimers and p50/ p65 heterodimers to importin ␣3. A, in vitro-translated p50, p65 or IB␣ proteins were allowed to bind to Sepharose-immobilized GSTimportins ␣1, ␣3, ␣5, ␣7, and ␤ for 1 h. Sepharose-bound proteins were dissolved in Laemmli sample buffer and separated on 10% SDS-PAGE. An autoradiogram of [ 35 S]methionine-labeled p50, p65, and IB␣ proteins is shown. In vitro translation products (c) are shown as controls. A similar gel was also stained with Coomassie Blue to visualize the amount of GST-importin isoforms. B, in vitro-translated p50, p65, or p50 ϩ p65 proteins were allowed to bind to Sepharose-immobilized GST-importin ␣3 as described above. The binding experiment was carried out in the absence (Ϫ) or presence of different amounts of in vitro-translated IB␣ protein as indicated. In vitro translation products (c) are shown as controls. Coomassie Blue-stained gel is shown to visualize the amount of GST-importin ␣3.
p50 binding whereas mutations in the arm repeats 7ϩ8 had no effect. To confirm the results obtained by cellular TNF-␣-stimulated NF-B proteins, we used in vitro translated p50 and p65 proteins and the arm repeat mutants in GST-importin ␣3 binding experiments. In this experiment also an arm 3ϩ8 mutated importin ␣3 was used. As is seen in Fig. 7D in vitro translated p50 and p65 gave identical results with the TNF-␣-stimulated cellular p50 and p65 proteins. Arm repeat 3 mutation in importin ␣3 prevented the binding of p50, and mutations in arm repeats 7ϩ8 prevented the binding of p65. Mutations in arm repeats 3ϩ8 in importin ␣3 prevented the binding of both proteins. These results show that the N-terminal arm repeats of importin ␣3 form the binding site for p50 whereas p65 is bound to the C-terminal NLS binding site.
NLS Defective p65 Does Not Have Any Effect on the Distribution of Cellular p50 -It has been suggested that p50 and p65 proteins have their own independently functioning NLSs (37,38). Here we have shown that these NLSs mediate p50 and p65 interactions with importin ␣3 (and importin ␣1) and regulate their nuclear import (Figs. 1, 2, 5, 6, and 7). Using confocal laser microscopy we compared the effects of transfected wildtype and NLS-mutated p65 protein on cellular location of p50. LMB was used to prevent the nuclear export of p65. When cells were transfected with wild-type or NLS-mutated p65 the distribution of the cellular p50 remained the same. p50 was mainly nuclear and showed a granular expression pattern both in TNF-␣-stimulated and in untreated cells (Fig. 8). Tran-siently transfected wild-type p65 was mainly cytoplasmic and after TNF-␣ stimulation it colocalized with p50 in the cell nucleus (Fig. 8). NLS-mutated p65 remained cytoplasmic also after TNF-␣ stimulation (Fig. 8). This suggests that a significant portion of p50 homodimers is transported into the nucleus independently of p65 and transport-deficient p65 (NLS Ϫ ) cannot function as a dominant negative for nuclear import of p50 under these circumstances.

DISCUSSION
Importin ␣3 and Importin ␣4 Are Novel Members of the NF-B Signal Transduction Pathway-Translocation of NF-B transcription factors from the cytoplasm into the nucleus is a critical step in their signal transduction pathway. NF-Bs contain classical NLS motifs, and they are imported into the nucleus via the importin ␣/␤ pathway. In the present study we show strong evidence that TNF-␣-activated NF-B p50/p65 heterodimers are transported into the nucleus preferentially by importin ␣3 and importin ␣4. Importin ␣3 also seems to be involved in the nuclear import of p50 homodimers in uninduced cells. It is shown that the previously identified NLSs of p50 and p65 molecules are the direct targets for importin ␣ binding.
In our study also bacterially expressed importin ␣1 bound to NF-B after TNF-␣ stimulation although to a lesser extent than importin ␣3. This binding was mediated by the NLSs of p50 and p65 proteins. However, no binding of baculovirus-expressed importin ␣1 to NF-B was detected under the same circumstances. Moreover, we did not observe any binding of cellular importin ␣1 to GST-p50 or GST-p65 proteins. On the basis of these findings, it cannot be predicted if importin ␣1 is specifically and significantly involved in NF-B nuclear transport.
Most importin ␣ isoforms are expressed in the same cells and tissues, but they may have distinct substrate specificities. It is likely that the whole NLS binding groove of importin ␣ contributes to the specificity of target protein binding, not only the few critical amino acids directly interacting with NLS. Similarly, the flanking sequences of the NLS contribute to the importin ␣ binding affinity and specificity. For example, SV40 T antigen NLS peptide binds to all importin isoforms (40 -45), whereas the full-length T antigen binds primarily to importin ␣3 and to a lesser extent to importin ␣1 (48).
Importin ␣ molecules have two NLS binding sites that directly interact with the NLS of the cargo. Arm repeats 2-4 comprise the N-terminal NLS binding site and arm repeats 7-9 the C-terminal NLS binding site. We have previously shown that importin ␣3 is able to use either its N-or C-terminal binding sites for binding different nucleus-targeted proteins. Influenza A virus nucleoprotein is bound to the C-terminal NLS binding site whereas simian virus 40 large T antigen is bound to the N-terminal NLS binding site (48). Our present study demonstrates another variation in theme, since NF-B p50 and p65 molecules are bound by different NLS binding sites of importin ␣3, p50 is bound by the N-terminal and p65 by the C-terminal NLS binding site.
It is an intriguing possibility that p50 and p65 NLSs may be bound simultaneously to the different NLS binding sites of the same importin ␣ molecule (Fig. 9). This kind of binding strategy may stabilize the complex during nuclear import. However, the question arises how p50 homodimers are bound to importin ␣ because p50 NLS seems to be bound only by the N-terminal binding site.
Importin ␣3 and ␣4 belong to the same importin subfamily and display a high degree of sequence homology (45) (Fig. 7, A  and B). We believe that importin ␣4 can function much in the same way in NF-B interactions as importin ␣3. It is likely that these importins also have distinct substrate specificities (45,64).
Like NF-B, signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors activated by cytokines. Upon activation STATs are phosphorylated, dimerized, and transported into the nucleus by the classical importin ␣/␤ pathway. In our previous study we showed that STAT1 and STAT2 contain nonclassical NLSs that become operative only in dimers. The NLSs of both partners of a STAT dimer have to be intact for nuclear import to take place (65). STAT1 homodimers and STAT1/STAT2 heterodimers bind to the C-terminal NLS binding site of importin ␣5 and two importin ␣ molecules are needed to transport the dimer into the nucleus (48,66). In contrast to STATs it seems that in NF-B dimers each NLS may function independently (37). However, it has also been suggested that both partners of a Rel homodimer require an intact NLS for proper nuclear import (67).
Are IB␣-bound NF-B Dimers Shuttling between the Cytoplasm and Nucleus?-Since p65 possesses both an NLS and a strong NES, IB free p50/p65 heterodimers and p65 homodimers are continuously transported into and out of the nucleus. In the presence of leptomycin B, an inhibitor of CRM1dependent nuclear export, p65 containing dimers accumulate in the nucleus (57)(58)(59). IB␣ contains its own unconventional NLS that is masked when the protein is bound to NF-B dimers (16,17,68). When IB␣ is not bound to NF-B dimers it can shuttle between the cytoplasm and the nucleus by means of its NLS and NES.
It has been suggested by several groups that in uninduced cells NF-B⅐IB␣ complexes continuously shuttle between the cytoplasm and the nucleus (56 -60). According to our results importin ␣3 does not bind to NF-B dimers when they are associated with IB␣. Our data suggest that NF-B and IB␣ are not transported into the nucleus as a complex. Our data rather support the previous findings that in resting cells cytoplasmic dissociation of the NF-B⅐IB␣ complex is followed by nuclear import of the single subunits rather than the complex as a whole (69,70). After re-association of NF-B and IB␣ in the nucleus, the complex is then transported back to the cytoplasm. This transport is mediated by IB␣ (56,57), or by the inherent NES of p65 (71).
We found that small amounts of p50 can associate with importin ␣3 under uninduced conditions. This form of p50 was free of IB␥ (the C-terminal-half of p105). In uninduced cells p105 is partially degraded, generating p50 (14,32,72,73). We assume that the basal binding of p50 to importin ␣3 detected is because of the continuous degradation of the p105, which genwere allowed to bind to Sepharose-immobilized wild type or arm repeat 3 or 7ϩ8 mutants of GST-importin ␣3 at ϩ4°C for 2 h. The binding experiments were carried out as described in the legend for Fig. 1. Western blots were stained with anti-p50 and anti-p65 antibodies as indicated. A similar gel was also stained with Coomassie Blue to visualize the amount of GST-importin ␣3. D, in vitro translated p65 (upper panel) and p50 (middle panel) or p50 ϩ p65 (lower panel) were allowed to bind to Sepharose-immobilized wild type or arm repeat 3, 7ϩ8, or 3ϩ8 mutants of GST-importin ␣3. Sepharose-bound proteins were dissolved in Laemmli sample buffer and separated on 10% SDS-PAGE. An autoradiogram of [ 35 S]methionine-labeled p50 and p65 proteins is shown. In vitro translation products (c) were used as controls. Coomassie Blue-stained gel is shown to visualize the amount of GST-importin ␣3.
FIG. 8. Colocalization of p50 with p65. HuH7 cells were transiently transfected with wild type or NLS mutant p65 gene constructs as indicated. The cells were left untreated or pretreated with LMB (2 ng/ml) for 20 min followed by stimulation with TNF-␣ (100 ng/ml) for 30 min in the presence of LMB (2 ng/ml). The cells were fixed and double stained with rabbit anti-p50 antibodies and mouse anti-Penta-His antibodies, followed by FITC-and Rhodamine Red-X-labeled secondary antibodies. p50 staining is shown as FITC-labeled (green) and p65 as Rhodamine Red-X-labeled (red). Colocalization is seen in yellow. Bar, 10 m.
In contrast to p50, we did not observe binding of p65 to importin ␣3 in uninduced cells. The formation of p50/p65 heterodimers is largely unknown but if p105 associates with p65, and is also degraded to some extent to generate p50/p65 heterodimers, why then did we not observe any uninduced binding of p50/p65 heterodimers to importin ␣3? It is possible that when IB␥ is degraded from the p65/p105 dimer the subsequent p50/p65 heterodimer rapidly binds IB␣ (or IB␤). After that, the continuous shuttling of p50/p65 may occur only after IB␣ dissociation is taking place (69,70). Nearly all of the studies on the accumulation of NF-B and IB␣ in the nucleus under uninduced conditions depend on the use of LMB for relatively long time periods. This indicates that the shuttling is a slow phenomenon and was not detectable in our GST-importin interaction experiments. Unlike p50/p65 heterodimers, p50 homodimers are not effectively regulated by IBs other than IB␥. Thus, the continuous degradation of p105 associated with p50 could provide the resting cell the small amount of p50 homodimers required. A conceptual model of NF-B transport into the nucleus is presented in Fig. 9.
The shuttling of the p50⅐p65⅐IB␣ complex has been suggested to be mediated by the free NLS of p50 (60). However, p50⅐p50⅐IB␥, p50⅐p65⅐IB␥, and p50⅐p65⅐IB␤ complexes do not shuttle between the cytoplasm and nucleus. It has been suggested that the cytoplasmic retention of these complexes could involve additional factors that mask their exposed NLSs (73). Alternatively, it is possible that when IB is bound to NF-B, importin molecules simply do not fit to interact with the free NLS.
Taken together, our data allow us to conclude that NF-B binds to importin ␣3 and importin ␣4 and is transported into the nucleus only when it is free of IB molecules. In the future, structural analysis of NF-B bound to importin molecules is needed to reveal the exact mechanism of interaction between these molecules. FIG. 9. Schematic model for constitutive nuclear import of p50 homodimers and TNF-␣-stimulated nuclear import of p50/p65 heterodimers. In uninduced cells p50/p105 heterodimers are constitutively processed to form p50 homodimers, which are transported into the nucleus. In TNF-␣-induced cells the p50/p65 heterodimer-bound IB␣ is phosphorylated by the IKK-complex and proteolytically degraded. Free p50 homodimers and p50/p65 heterodimers are bound to importin ␣ and consequently imported into the nucleus. IB␣ is shown in purple, NLSs in red, p65 NES in yellow, and importin ␣ in orange.