In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.     

The transport mechanism of metallothionein is different from that of classical NLS-bearing protein

A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin alpha and importin beta, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjugated allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-binding was observed in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addition of an excess amount of unlabeled MT. Different elution profiles were observed for the localization-promoting activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was determined to be a wheat germ agglutinin-insensitive, GTPgammaS-sensitive, and anti-Ran antibody-sensitive process. Green fluorescent protein-metallothionein (GFP-MT) fusion protein was also localized in the nucleus in the stable transformant of CHL-IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization.     

O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.     

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.     

Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells

Mediated transport across the nuclear envelope was investigated in proliferating and growth-arrested (confluent or serum starved) BALB/c 3T3 cells by analyzing the nuclear uptake of nucleoplasmin-coated colloidal gold after injection into the cytoplasm. Compared with proliferating cells the nuclear uptake of large gold particles (110-270 A in diameter, including the protein coat) decreased 5.5-, 33-, and 78- fold, respectively, in 10-, 14-17-, and 21-d-old confluent cultures; however, the relative uptake of small particles (total diameter 50-80 A) did not decrease with increasing age of the cells. This finding suggests that essentially all pores remain functional in confluent populations, but that most pores lose their capacity to transport large particles. By injecting intermediate-sized gold particles, the functional diameters of the transport channels in the downgraded pores were estimated to be approximately to 130 and 110 A, in 14-17- and 21-d- old cultures, respectively. In proliferating cells, the transport channels have a functional diameter of approximately 230 A. The mean diameters of the pores (membrane-to-membrane distance) in proliferating and confluent cells (728 and 712 A, respectively) were significantly different at the 10%, but not the 5%, level. No differences in pore density (pore per unit length of membrane) were detected. Serum- deprived cells (7-8 d in 1% serum or 4 d in 0.5% serum) also showed a significant decrease in the nuclear uptake of large, but not small, gold particles. Thus, the permeability effects are not simply a function of high cell density but appear to be growth related. The possible functional significance of these findings is discussed.     

Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.     

Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0°C and is Ca²⁺-independent. By contrast, translocation requires ATP hydrolysis, Ca²⁺, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells. © 1996 Wiley-Liss, Inc.     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts.

In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.     

Role of nuclear pore complex in simian virus 40 nuclear targeting.

Cytoplasmically injected simian virus 40 (SV40) virions enter the nucleus through nuclear pore complexes (NPCs) and can express large T antigen shortly thereafter (J. Clever, M. Yamada, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 88:7333-7337, 1991). The nuclear import of the protein components of introduced SV40 was reversibly arrested by chilling and energy depletion, corroborating our previous observation that the nuclear entry of injected SV40 is blocked in the presence of wheat germ agglutinin and an antinucleoporin monoclonal antibody (mAb414), general inhibitors of NPC-mediated import. The nuclear accumulation of virion protein components and large T antigen in nonpermissive NIH 3T3 cells was similar to that in the permissive host, indicating that the ability to use NPCs as a route of nuclear entry appears to be a general property of the injected virus. Injected virions were capable of completing their lytic cycle and forming plaques in permissive cells. During the early phase of SV40 infection, the cytoplasmic injection of mAb414 effectively blocked nuclear T-antigen accumulation for up to 8 h of infection but had very little effect after 12 h of infection. The time-dependent interference with nuclear T-antigen accumulation by the antinucleoporin antibody is consistent with the hypothesis that the infecting virions enter the nucleus through NPCs. The interference study also suggests that the early phase of infection consists of at least two steps: a step for virion cell entry and intracytoplasmic trafficking and a step for virion nuclear entry followed by large-T-antigen gene expression and subsequent nuclear localization of the gene product. Virions were visualized as electron-dense particles in ultrathin sections of samples in which transport was permitted or arrested. In the former cells, electron-dense particles were predominantly observed in the nucleus. The virions were distributed randomly and nonuniformly in the nucleoplasm but were not observed in heterochromatin or in nucleoli. In the latter cells, the electron-dense particles were seen intersecting the nuclear envelope, near the inner nuclear membrane, and in NPCs. In tangential cross sections of NPCs, which appeared as donut-shaped structures, a spherical electron-dense particle was observed in the center of the structure. Immunoelectron microscopy revealed that NPCs were selectively decorated with 5-nm colloidal gold particles-anti-Vp1 immunoglobulin G at the cytoplasmic entrance to and in NPCs, confirming that the morphologically observed electron-dense particles in NPCs contain the viral structural protein. These results support the hypothesis that the nuclear import of SV40 is catalyzed through NPCs by an active transport mechanism that is similar to that of other karyophiles.     

Cytosolic import factor- and Ran-independent nuclear transport of ribosomal protein L5

Ribosomal protein L5 is a shuttling protein that, in Xenopus oocytes, is involved in the nucleocytoplasmic transport of 5S rRNA. As demonstrated earlier, L5 contains three independent nuclear import signals (NLSs), which function in oocytes as well as in somatic cells. Upon physical separation, these NLSs differ in respect to their capacity to bind to nuclear import factors in vitro and to mediate the nuclear import of a heterologous RNP in vivo. As reported in this communication, analysis of the in vitro nuclear import activity of these three NLSs reveals that they also differ in respect to their requirements for cytosolic import factors and Ran. Nuclear import mediated by the N-terminal and the central NLS depends on cytosolic import factor(s) and Ran, whereas import via the C-terminal NLS occurs independently from these factors. Thus, the presence of multiple NLSs in ribosomal protein L5 appears to allow for efficient nuclear transport via utilisation of multiple, mechanistically different import pathways.     

Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.

Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.     

The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.     

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.     

Ceramide regulation of nuclear protein import

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.     

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)