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Ikappa B Family Members Function by Different Mechanisms*

Winnie F. Tam and Ranjan SenDagger

From the Rosenstiel Basic Medical Sciences Research Center and the Department of Biology, Brandeis University, Waltham, Massachusetts 02454

Received for publication, December 22, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Ikappa B family of proteins regulates NF-kappa B-dependent transcription by inhibiting DNA binding and localizing these factors to the cell cytoplasm. Ikappa Balpha does this by shifting the balance between nuclear import of Rel proteins and their export from the nucleus. Here we show that, unlike Ikappa Balpha , Ikappa Bbeta and Ikappa Bepsilon appear to sequester p65 or c-Rel in the cytoplasm by inhibiting nuclear import. Furthermore, because Ikappa Bbeta does not undergo nucleocytoplasmic shuttling, it cannot remove nuclear proteins like Ikappa Balpha does. We conclude that the mechanism of action differs among Ikappa B family members.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The NF-kappa B1/Rel family of transcription factors plays a central role in immune and inflammatory responses (1). In most cell types these proteins are sequestered in the cell cytoplasm complexed to a family of inhibitory Ikappa B proteins (2, 3). Cellular activation results in Ikappa B degradation, which leaves the DNA-binding protein free to translocate to the nucleus and activate gene expression. Because of the widespread effects of NF-kappa B activation, its localization in the cytoplasm must be strictly maintained. Ikappa Balpha -deficient mice are a striking example of the importance of NF-kappa B sequestration in the cytoplasm; these mice die of a wasting disease that has been attributed to tumor necrosis factor-alpha production (4, 5). Nuclear factor-kappa B has also been detected in several diseased tissues, where it has been proposed to contribute to the pathology in part by inhibiting apoptosis (6, 7).

The association of Rel with Ikappa Balpha has been proposed to hide the NLS of Rel proteins (8, 9), thereby precluding nuclear entry of the transcription factor. In addition to hiding the NLS, association with Ikappa Balpha also inhibits DNA binding by NF-kappa B. Thus, association of NF-kappa B with Ikappa B ensures that NF-kappa B-dependent gene transcription occurs only when cells are stimulated appropriately.

Recently, Ikappa Balpha has been shown to shuttle between the nucleus and the cytoplasm. Nuclear entry is mediated by a nonclassical NLS located in the second ankyrin repeat of Ikappa Balpha (10-12), and nuclear export is determined by a CRM1-dependent nuclear export sequence located in the N-terminal domain preceding the first ankyrin domain (13-15). A second nuclear export sequence has been identified at the C terminus of Ikappa Balpha , but its functional significance is unclear at present (16, 17). The observation that cytoplasmic sequestration of p65·RelA also required nuclear export was unexpected and led to a reassessment of the existing sequestration model. We and others (13-15) have proposed that the cytoplasmic location of Rel proteins by Ikappa Balpha is a dynamic process that depends on the active export of Rel·Ikappa Balpha complexes out of the nucleus.

In our earlier studies we also showed that Ikappa Bbeta and Ikappa Bepsilon , unlike Ikappa Balpha , do not shuttle via the CRM1 pathway. Specifically, the subcellular distribution of GFP-Ikappa Bbeta or GFP-Ikappa Bepsilon in yeast was not affected by a mutation in the CRM1 gene, and leptomycin B (LMB) treatment did not alter the location of these proteins in transiently transfected mammalian cells. In addition, we did not detect an association between CRM1 protein and either Ikappa Bbeta or Ikappa Bepsilon in a yeast two-hybrid assay (15). In this paper we demonstrate that cytoplasmic retention of Rel proteins by Ikappa Bbeta and Ikappa Bepsilon involves sequestration rather than tilting the balance of nuclear import and export as is the case with Ikappa Balpha . Furthermore, although newly synthesized Ikappa Bbeta can enter the nucleus, it cannot restore nuclear Rel proteins to the cytoplasm. These observations suggest that Ikappa Bbeta and Ikappa Bepsilon function differently from Ikappa Balpha .


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Strains-- D5 h3 T hybridoma cells and A20 mature B cells were grown in Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively, with 10% heat-inactivated fetal bovine serum, 50 µM beta -mercaptoethanol and antibiotics. COS cells were cultured in Dulbecco's modified Eagle's medium with 10% newborn calf serum and antibiotics. Yeast strain W303 and its transformants were generally grown in synthetic medium with the appropriate amino acid and nitrogen base supplement.

Plasmids-- pGFP-p65 and pCDNA3-HA-Ikappa Balpha have been described previously (15). pGFP-cRel contains full-length murine cRel in frame after GFP. pCDNA3-Myc-Ikappa Bbeta and pCDNA3-Myc-Ikappa Bepsilon were made by inserting full-length murine Ikappa Bbeta and Ikappa Bepsilon cDNA, respectively, in frame behind a c-Myc tag (MEQKLISEEDL). Yeast galactose-inducible plasmid encoding GFP-p65 and copper-inducible HA-Ikappa Balpha (pCuIkappa Balpha ) have been described previously (15). The copper-inducible HA-Ikappa Bbeta was made by replacing the Ikappa Balpha gene with a murine Ikappa Bbeta full-length gene in the same vector. All plasmids used in this study were confirmed by sequencing, and expression of proteins was verified by immunoblotting.

Immunostaining-- The procedures for immunostaining adherence cells were the same as described previously (15). For staining suspension cells (T and B cells), the procedures were also as described previously (18).

Protease Digestion-- The proteases Asp-N and Lys-C were purchased from Roche Molecular Biochemicals. Proteases were used according to the manufacturers' specifications.

Fluorescence Microscopy-- The subcellular localization of GFP and the immunofluorescence signals were observed by fluorescence microscopy (Axiophot II, Zeiss) with a GFP generic filter, fluorescein isothiocyanate, rhodamine, and DAPI filter.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nuclear export property of Ikappa Balpha is essential for cytoplasmic location of Rel proteins. However, Ikappa Bbeta and Ikappa Bepsilon , which are not nucleo-cytoplasmic shuttling proteins, can also effectively localize Rel proteins to the cytoplasm. One possibility was that cytoplasmic retention by Ikappa Bbeta /epsilon may be mediated by export determinants in the Rel proteins. To test this possibility, we coexpressed green fluorescent protein (GFP)-tagged Rel proteins with Ikappa Bbeta or Ikappa Bepsilon in COS cells and assayed the location of Rel proteins by GFP fluorescence. Both p65 and c-Rel (data not shown) were located in the cytoplasm in the presence of Ikappa Bbeta (Fig. 1A, left panel). However, these complexes did not translocate to the nucleus when the cells were treated with LMB, an inhibitor of CRM1-mediated nuclear export (Fig. 1A, right panel). Therefore, CRM1 was not involved in determining the subcellular location of these complexes. Similar results were obtained with Ikappa Bepsilon . As expected, Ikappa Balpha -associated p65, or c-Rel (data not shown), was predominantly nuclear in LMB-treated cells (Fig. 1A, top row). Thus, cytoplasmic retention by Ikappa Bbeta and Ikappa Bepsilon may involve true sequestration rather than a balance between import and export as is the case with Ikappa Balpha .



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Fig. 1.   Rel·Ikappa Balpha complexes shuttle continuously, but Rel·Ikappa Bbeta complexes do not. A, GFP-p65 was transiently transfected with HA-Ikappa Balpha , Myc-Ikappa Bbeta , or Myc-Ikappa Bepsilon into COS cells. Half the cells were then treated with LMB (10 ng/ml) for 3 h. Untreated (left panel) or LMB-treated cells (right panel) were fixed for fluorescent visualization. Green fluorescence shows a GFP-p65 signal. The red fluorescence shows an Ikappa B signal from rhodamine-conjugated antibodies against either the HA tag or the Myc tag. Blue fluorescence shows DAPI staining of nuclei. B, A20 B cells were settled on specially treated coverslips (Fisher). Half of these were treated with LMB (100 ng/ml) for 45 min. Cells with or without LMB treatment were fixed and permeabilized for immunostaining. Green fluorescence shows endogenous Ikappa Balpha (first row) and Ikappa Bbeta (second row) detected by fluorescein isothiocyanate-conjugated antibodies against anti-Ikappa Balpha and anti-Ikappa Bbeta respectively. Representative results are shown from one of three independent experiments.

These observations were confirmed in mammalian cells by investigating the shuttling dynamics of endogenous Rel·Ikappa B complexes. Endogenous proteins in mature B (A20) and mature T (D5 h3) cell lines (data not shown) were visualized by staining fixed, permeabilized cells with anti-Ikappa Balpha , or anti-Ikappa Bbeta , antibodies in the presence or absence of LMB to block nuclear export. In untreated cells both Ikappa Bs were predominantly cytoplasmic (Fig. 1B, left panel). A 1-h LMB treatment induced considerable nuclear translocation of Ikappa Balpha but not Ikappa Bbeta (Fig. 1B, right panel). Because most of the cellular Ikappa B is associated with Rel proteins, we concluded that Rel·Ikappa Balpha complexes shuttled continuously, but Rel·Ikappa Bbeta complexes did not. Lack of Rel·Ikappa Bbeta shuttling is consistent with sequestration being the major mechanism of cytoplasmic retention by Ikappa Bbeta .

We found more direct evidence for differences in interaction between Ikappa Balpha or Ikappa Bbeta and p65 through partial proteolysis assays. p65 protein was expressed by transient transfection in BOSC 23 cells in the presence of HA-Ikappa Balpha or Myc-Ikappa Bbeta . The p65·Ikappa B complex was immunoprecipitated from whole cell extract with anti-Ikappa Balpha antibody or anti-Ikappa Bbeta antibody and digested with different proteases. The p65 fragments were detected using antibodies directed against the N or C terminus of p65 to estimate the cut site from one or the other end of p65. Only two of seven proteases showed significant differences in the pattern of p65 fragments generated in the presence of Ikappa Balpha or Ikappa Bbeta .

p65 alone generated one major fragment when treated with Asp-N of ~28 kDa when assayed from the C terminus (Fig. 2, lanes 5 and 6); this corresponds to a cut site located 293 amino acids from the N terminus (Fig. 2, top). In the p65·Ikappa Balpha complex, two bands of approximately equal intensity were seen (Fig. 2, lanes 1 and 2), whereas in the p65·Ikappa Bbeta complex the faster mobility (23 kDa) band was enhanced. Therefore, cutting at residue 293 was reduced in the p65·Ikappa Balpha complex compared with p65 alone, allowing the detection of the cut site at residue 360 (which was not evident with p65 alone). This is presumably because of the protection of the p65 NLS by Ikappa Balpha , which lies close to residue 293 between residues 301 and 304. Cutting at 293 was further inhibited in the p65·Ikappa Bbeta complex as shown by a relative increase in the intensity of the 23-kDa compared with the 28-kDa band. These observation suggest that the region around residue 293, including the NLS, is more protected in the p65·Ikappa Bbeta complex.



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Fig. 2.   Protease digestion of p65·Ikappa Balpha and p65·Ikappa Bbeta complexes. p65 protein was transiently transfected in BOSC 23 cells alone (lane 1, 2, 7, and 8), in the presence of HA-Ikappa Balpha (lane 3, 4, 9, and 10) or Myc-Ikappa Bbeta (lane 5, 6, 11, and 12). Anti-Ikappa Balpha or anti-Ikappa Bbeta antibodies were used to immunoprecipitate the p65·Ikappa B complex from whole cell extracts. Precipitated materials were digested with Asp-N (left panel, A) and Lys-C (right panel, L). Undigested samples are indicated by a "-" in the figures. Digested and undigested products were fractionated by SDS-polyacrylamide gel electrophoresis and detected using an antibody against the C terminus of p65 (left panel) or antibody against the N terminus of p65 (right panel). Relevant protease sites of p65 were predicted by MacVector version 6.0 (top panel). NLS represents the nuclear localization signal of p65, with critical residues between residues 301 and 304. Arrows (in the lower panels) indicate the relative degree of Lys-C or Asp-N cutting in the Ikappa Bbeta ·p65 and Ikappa Balpha ·p65 complexes.

p65·Ikappa Balpha and p65·Ikappa Bbeta complexes were also probed using the protease Lys-C and p65 antibodies directed against the N terminus. Increased cutting at the residue 425 site was evident in the Ikappa Bbeta complex compared with the Ikappa Balpha complex (Fig. 2, lanes 8 and 11). These observations also support the interpretation that Ikappa Balpha and Ikappa Bbeta interact differently with p65. We suggest that the p65 NLS is better hidden by Ikappa Bbeta than by Ikappa Balpha .

The simplest interpretation of the experiments described above was that Rel·Ikappa Bbeta complexes did not enter the nucleus because the nuclear localization sequences in both proteins were very effectively hidden in the complex. Therefore, the question of nuclear export did not arise. However, the question remained that if any Rel·Ikappa Bbeta complexes formed in the nucleus, would Ikappa Bbeta be able to bring the complex out to the cytoplasm? Such a situation may occur at the end of cell stimulation when Rel proteins are already nuclear and new Ikappa Bs are synthesized to terminate NF-kappa B-dependent gene expression. We addressed this question in a yeast model.

We have previously shown that export-dependent cytoplasmic localization of p65 by Ikappa Balpha can be recapitulated in yeast (15). To test the properties of Ikappa Bbeta , we coexpressed Ikappa Bbeta and GFP-p65 from galactose-inducible promoters in wild type or Crm1p-deficient (crm1-1) yeast strains. GFP-p65 was located in the cytoplasm under these conditions in both strains (data not shown), correlating closely with the observations in mammalian cells (Fig. 1). In contrast, when GFP-p65 and Ikappa Balpha were coexpressed in crm1-1 cells, the complex remained in the nucleus (15). To compare the ability of Ikappa Balpha and Ikappa Bbeta to remove nuclear p65, we expressed GFP-p65 using a galactose-inducible promoter, followed by either Ikappa Balpha , or Ikappa Bbeta , from a copper-inducible promoter. A 3-h induction with galactose was followed by growth in glucose to suppress GFP-p65 transcription. In cells that did not contain Ikappa B expression vectors, GFP fluorescence was strictly nuclear. Even when cells contained either Ikappa Balpha or Ikappa Bbeta expression plasmids, GFP fluorescence was largely restricted to the nucleus, although whole cell expression was observed in ~15% of the cells (Fig. 3, middle and bottom rows, left panel). Cytoplasmic expression under these conditions was most likely due to basal Ikappa Balpha , or Ikappa Bbeta , expression from the copper-inducible promoter. Induction of Ikappa Balpha with copper for 2 h resulted in a significant redistribution of GFP-p65 to the cytoplasm, indicating that the newly synthesized Ikappa Balpha exported nuclear GFP-p65 to the cytoplasm (Fig. 3, middle row, right panel). This was mediated by Crm1p because it did not occur in the crm1-1 strain that contains a mutated CRM1 gene (data not shown). In contrast, there was little redistribution of GFP-p65 after secondary induction of Ikappa Bbeta (Fig. 3, bottom row, right panel). The small increase in whole cell GFP-p65 expression was probably because of residual GFP-p65 translation during Ikappa Bbeta induction, which resulted in its cytoplasmic sequestration. These observations indicate that Ikappa Bbeta cannot remove nuclear Rel proteins to the cytoplasm.



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Fig. 3.   Sequential induction of GFP-p65 and Ikappa B. GFP-p65 was cloned into an expression plasmid with a galactose-inducible promoter. HA-tagged Ikappa Balpha , or Ikappa Bbeta , was cloned into an expression plasmid containing a copper-inducible promoter. Yeast strain W303 transformed with both GFP-p65 and HA-Ikappa B expression plasmids was treated with galactose for 3 h to induce GFP-p65 expression (left panel). Half of the cells were then treated with glucose to terminate the expression of GFP-p65 followed by 0.75 mM copper sulfate to induce Ikappa B expression (right panel). GFP fluorescence was visualized directly with fluorescence microscopy. Whole cell extracts were made from the cells to confirm the induction of GFP-p65, HA-Ikappa Balpha , and HA-Ikappa Bbeta proteins by immunoblotting (data not shown). Results shown are from one of three independent experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found that p65 or c-Rel associated with Ikappa Bbeta or Ikappa Bepsilon were retained in the cytoplasm, although these Ikappa Bs did not shuttle via the CRM1 pathway. We suggest that Ikappa Bs, unlike Ikappa Balpha , sequester rather than shuttle Rel proteins, which implies that there is no available NLS in the Rel·Ikappa Bbeta (or Ikappa Bepsilon ) complexes to induce nuclear entry. Conversely, Rel·Ikappa Balpha complexes must have an available NLS to shuttle. We hypothesize that the Rel and not the Ikappa B component provides the functional NLS of a Rel·Ikappa B complex. Thus, Ikappa Bbeta or Ikappa Bepsilon must hide the Rel NLS more effectively than Ikappa Balpha . Evidence in favor of this idea was obtained from partial proteolytic studies of p65·Ikappa B complexes.

The sequestration mechanism is based on the lack of an effect of leptomycin B or a mutated CRM1 gene in Rel protein localization by Ikappa Bbeta . Alternatively, these results could indicate that Rel·Ikappa Bbeta complexes shuttled by a CRM1-independent pathway. To test this theory, we generated nuclear Rel·Ikappa Bbeta complexes and determined whether they could reach the cytoplasm by an unidentified pathway. As shown in Fig. 3, Ikappa Bbeta -mediated GFP-p65 export was inefficient compared with Ikappa Balpha . We conclude that Ikappa Bbeta is not an export chaperone like Ikappa Balpha . Consequently, Ikappa Bbeta cannot efficiently down-regulate nuclear Rel proteins to restore the resting state of the cell. These results highlight the functional differences between Ikappa Balpha and Ikappa Bbeta .

Cheng et al. (19) showed that substituting Ikappa Bbeta for the Ikappa Balpha gene compensated for the most obvious defects in Ikappa Balpha -/- mice. They concluded that Ikappa Balpha and Ikappa Bbeta were functionally similar and that regulation of expression accounted for most of the phenotype of Ikappa Balpha -deficient mice. Our contrasting conclusion regarding the mechanism of Ikappa Balpha and Ikappa Bbeta function is not at odds with the biological results. Clearly, if sufficient Ikappa Bbeta is synthesized in a cell, it can retain Rel proteins in the cytoplasm, albeit by a mechanism different from Ikappa Balpha . The biological results show that retention of Rel proteins by either mechanism is good enough to rescue lethality. That Ikappa Bbeta is a less efficient nuclear export chaperone than Ikappa Balpha may be manifest under conditions that were not directly assayed, such as during an immune response or chronic inflammation. We suggest that control of such situations may require the active export-dependent reduction of NF-kappa B activity.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Rosenstiel Basic Medical Sciences Research Ctr., Brandeis University, 415 South St., Waltham, MA 02454. E-mail: sen@brandeis.edu.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C000916200


    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor-kappa B; NLS, nuclear localization signal; GFP, green fluorescent protein; LMB, leptomycin B; HA, hemagglutinin; DAPI, 4,6-diamidino-2-phenyllindole.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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