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κB-Ras Binds to the Unique Insert within the Ankyrin Repeat Domain of IκBβ and Regulates Cytoplasmic Retention of IκBβ·NF-κB Complexes

2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês

10.1074/jbc.m301021200

1083-351X

Yi Chen, Joann Wu, Gourisankar Ghosh,

Cytokine Signaling Pathways and Interactions

The IκBα and IκBβ proteins inhibit the transcriptional potential of active NF-κB dimers through stable complex formation. It has been shown that inactive IκBα·NF-κB complexes shuttle in and out of the nucleus, whereas IκBβ·NF-κB complexes are retained exclusively in the cytoplasm of resting cells. The biochemical mechanism underlying this functional difference and its consequences are unknown. Although the two IκB proteins are significantly homologous, IκBβ contains a unique 47-amino acid insertion of unknown function within its ankyrin repeat domain. In this study, we assess the role of the IκBβ insert in regulating cytoplasmic retention of IκBβ·NF-κB complexes. Deletion of the IκBβ insert renders IκBβ·NF-κB complexes capable of shuttling between the nucleus and cytoplasm, similar to IκBα·NF-κB complexes. A small Ras-like G-protein, κB-Ras, participates with the IκBβ insert to effectively mask the NF-κB nuclear localization potential. Similarly, a complex between NF-κB and a mutant IκBβ protein containing four serine to alanine mutations within its C-terminal proline, glutamic acid, serine, and threonine-rich sequence exhibits nucleocytoplasmic shuttling. This suggests a phosphorylation state-dependent role for the C-terminal proline, glutamic acid, serine, and threonine-rich sequence of IκBβ in proper localization of IκBβ·NF-κB complexes. These results are consistent with structural studies, which predicted that binary IκBβ·NF-κB complexes should be capable of nuclear translocation, and with previous observations that hypophosphorylated IκBβ·NF-κB complexes can reside in the nucleus. The IκBα and IκBβ proteins inhibit the transcriptional potential of active NF-κB dimers through stable complex formation. It has been shown that inactive IκBα·NF-κB complexes shuttle in and out of the nucleus, whereas IκBβ·NF-κB complexes are retained exclusively in the cytoplasm of resting cells. The biochemical mechanism underlying this functional difference and its consequences are unknown. Although the two IκB proteins are significantly homologous, IκBβ contains a unique 47-amino acid insertion of unknown function within its ankyrin repeat domain. In this study, we assess the role of the IκBβ insert in regulating cytoplasmic retention of IκBβ·NF-κB complexes. Deletion of the IκBβ insert renders IκBβ·NF-κB complexes capable of shuttling between the nucleus and cytoplasm, similar to IκBα·NF-κB complexes. A small Ras-like G-protein, κB-Ras, participates with the IκBβ insert to effectively mask the NF-κB nuclear localization potential. Similarly, a complex between NF-κB and a mutant IκBβ protein containing four serine to alanine mutations within its C-terminal proline, glutamic acid, serine, and threonine-rich sequence exhibits nucleocytoplasmic shuttling. This suggests a phosphorylation state-dependent role for the C-terminal proline, glutamic acid, serine, and threonine-rich sequence of IκBβ in proper localization of IκBβ·NF-κB complexes. These results are consistent with structural studies, which predicted that binary IκBβ·NF-κB complexes should be capable of nuclear translocation, and with previous observations that hypophosphorylated IκBβ·NF-κB complexes can reside in the nucleus. NF-κB is a family of inducible, dimeric transcription factors that activate the expression of genes involved in the immune response and inflammation, development, and apoptosis (1Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5515) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4550) Google Scholar, 3Gilmore T.D. Oncogene. 1999; 18: 6842-6844Crossref PubMed Scopus (345) Google Scholar). 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Chem. 2000; 275: 24847-24856Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 25Venkatakrishnan A. Stecenko A.A. King G. Blackwell T.R. Brigham K.L. Christman J.W. Blackwell T.S. Am. J. Respir. Cell Mol. Biol. 2000; 23: 396-403Crossref PubMed Scopus (154) Google Scholar). In each case, IκBβ has been shown to be the critical mediator of persistent NF-κB activity. The mechanism underlying this persistently active NF-κB remains unclear. In an effort to understand the source of differential NF-κB activation kinetics, several groups have begun investigating the molecular mechanisms of action of the IκBα and IκBβ inhibitor proteins. One clear difference has emerged involving the manner by which these two proteins regulate NF-κB subcellular localization (26Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3260) Google Scholar). Immunofluorescence studies have revealed that IκBα·NF-κB complexes shuttle between the cytoplasm and nucleus in quiescent cells (27Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 28Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Crossref PubMed Scopus (318) Google Scholar, 29Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Crossref PubMed Scopus (133) Google Scholar). In contrast, inactive IκBβ·NF-κB complexes remain exclusively in the cell cytoplasm (30Huang T.T. Miyamoto S. Mol. Cell. Biol. 2001; 21: 4737-4747Crossref PubMed Scopus (89) Google Scholar, 31Tam W.F. Sen R.J. J. Biol. Chem. 2001; 276: 7701-7704Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). A mechanistic explanation for the dynamic shuttling behavior of IκBα·NF-κB complexes has been described recently. X-ray structures of the IκBα·NF-κB p50/p65 heterodimer complex revealed that IκBα masks the nuclear localization signal (NLS) 1The abbreviations used are: NLS, nuclear localization signal; PEST, proline, glutamic acid, serine, and threonine-rich sequence; LMB, leptomycin B; HA, hemagglutinin. 1The abbreviations used are: NLS, nuclear localization signal; PEST, proline, glutamic acid, serine, and threonine-rich sequence; LMB, leptomycin B; HA, hemagglutinin. of the NF-κB p65 subunit but fails to mask the p50 subunit NLS (33Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 34Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). Protease protection assays revealed that one NLS in the IκBα·NF-κB p65 homodimer and IκBα·NF-κB c-Rel homodimer complexes is also exposed to solvent and sensitive to protease cleavage (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 2C. Phelps, unpublished data. 2C. Phelps, unpublished data. Nuclear import of the IκBα·NF-κB p50/p65 heterodimer complex is imparted by the free p50 NLS, whereas the one free p65 subunit NLS is sufficient to convey similar shuttling properties upon the IκBα·NF-κB p65 homodimer complex (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Active export of the complex from the nucleus relies on nuclear export signals located in the N-terminal signal response region of IκBα and the transactivation domain of the p65 subunit (27Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 28Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Crossref PubMed Scopus (318) Google Scholar, 29Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Crossref PubMed Scopus (133) Google Scholar, 35Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Crossref PubMed Scopus (86) Google Scholar). In an accompanying study, we describe the x-ray crystal structure of an IκBβ·NF-κB p65 homodimer complex (see Ref. 43Malek S. Huang D.-B. Huxford T. Ghosh S. Ghosh G. J. Biol. Chem. 2003; 278: 23094-23100Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The structure reveals that, like IκBα·NF-κB complexes, the NLS of one NF-κB p65 subunit (subunit A) is effectively masked by IκBβ, whereas the second p65 subunit (subunit B) NLS is largely solvent-exposed. This observation is somewhat unexpected because the second NF-κB subunit NLS is significantly less sensitive to cleavage by proteases when bound to IκBβ (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, it remains unclear as to how IκBβ·NF-κB complexes are retained within the cytoplasm of resting cells. Recently, a small Ras-like protein was identified from a yeast two-hybrid screen as an IκBβ C-terminal PEST sequence-interacting factor and inhibitor of NF-κB activation (36Fenwick C. Na S.Y. Voll R.E. Zhong H. Im S.Y. Lee J.W. Ghosh S. Science. 2000; 287: 869-873Crossref PubMed Scopus (88) Google Scholar). It was suggested that this protein, named κB-Ras, might function by inhibiting IκBβ degradation. In the present study, we show that κB-Ras is a critical regulator of IκBβ·NF-κB complex subcellular distribution. κB-Ras binds through a unique 47-amino acid insert between ankyrin repeats 3 and 4 of IκBβ and partially masks one p65 subunit NLS in vitro and completely masks it in vivo. The conversion of serines in the IκBβ PEST sequence to non-phosphorylatable residues further alters the subcellular localization properties of IκB·NF-κB complexes. These data suggest that additional factors may bind κB-Ras and IκBβ·NF-κB complexes and sequester them to the cytoplasm of quiescent cells. We propose that regulated association and dissociation of κB-Ras determine the subcellular localization of IκBβ·NF-κB complexes. Mammalian Cell Transfection—HeLa cell transfection was performed by the LipofectAMINE method (Invitrogen). Leptomycin B (LMB) (5 ng/ml) was added 3 h before harvesting cells. LMB is a generous gift from Prof. M. Yoshida (University of Tokyo, Tokyo, Japan). Immunofluorescence—Cells (with or without LMB treatment) were fixed in 3% paraformaldehyde for 20 min at room temperature and then permeabilized with phosphate-buffered saline buffer containing 0.5% Nonidet P-40 and 0.01% sodium azide (ISB). Blocking was done using 5 mg/ml bovine serum albumin followed by incubation for 30 min with primary antibodies in ISB. Cells were then washed three times with ISB. Fluorescent-tagged secondary antibody was added in ISB at room temperature. Immunoprecipitation and Western Analysis—Cells were washed three times in phosphate-buffered saline buffer. Cytoplasmic extracts were made by lysing cells in 1% Triton X-100, 20 mm Tris-HCl (pH 7.6), 200 mm NaCl, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride (lysis buffer). Fifty μg of extract was mixed with protein A-agarose and primary antibodies and incubated at 4 °C overnight. The immunoprecipitates were washed three times in lysis buffer and eluted with SDS-PAGE buffer by heating at 100 °C for 5 min. The supernatant was separated by10% SDS-PAGE. The separated proteins in the gel were transferred to Hybond nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% milk in phosphate-buffered saline with 0.2% Tween and incubated with anti-p65 polyclonal antibody (H-286; Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit Ig (Santa Cruz Biotechnology). Blots were visualized by use of the ECL reagent kit (Amersham Biosciences). For in vitro immunoprecipitation experiments, 0.5 μg of NF-κB was mixed with 4 μg of IκB in the presence or absence of 4 μg of κB-Ras in a 15 μl binding reaction. The mixture was incubated on ice for 2 h followed by dilution to 100 μl in lysis buffer. Four μl of this diluted complex was used for the immunoprecipitation reaction using 0.1 μg of the anti-p65 NLS monoclonal antibody (a generous gift from Roche Diagnostics). The immunoprecipitates were then loaded on a Western blot, as described above. The Unique IκBβ Insert Inhibits Nuclear Import of IκBβ·NF-κB Complexes—The finding that one p65 subunit (subunit B) NLS is at most weakly bound by IκBβ suggests that this NLS may also be capable of translocating the IκBβ·NF-κB complex to the nucleus. It has been shown previously that IκBβ·NF-κB complexes are cytoplasmic in quiescent cells (30Huang T.T. Miyamoto S. Mol. Cell. Biol. 2001; 21: 4737-4747Crossref PubMed Scopus (89) Google Scholar, 31Tam W.F. Sen R.J. J. Biol. Chem. 2001; 276: 7701-7704Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, it follows that in quiescent cells the p65 subunit B NLS polypeptide must bind to IκBβ more stably than the binding mode revealed in IκBβ·NF-κB p65 homodimer complex crystal structure (see Ref. 43Malek S. Huang D.-B. Huxford T. Ghosh S. Ghosh G. J. Biol. Chem. 2003; 278: 23094-23100Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). To test whether the unique 47-amino acid insertion within the ankyrin repeat domain of IκBβ plays any role in determining the subcellular localization of IκBβ·NF-κB complexes, we deleted this insert from IκBβ (residues 152–192) and observed the co-localization of this mutant (IκBβΔ-(152–192)) with NF-κB p65 homodimer in HeLa cells (Fig. 1A). We observe that both complexes are cytoplasmic in resting cells. However, in the presence of LMB, an inhibitor of nuclear export receptor CRM1, the mutant IκBβΔ-(152–192)·NF-κB p65 homodimer complex, but not the wild type complex, is predominantly nuclear (37Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar, 38Kudo N. Khochbin S. Nishi K. Kitano K. Yanagida M. Yoshida M. Horinouchi S. J. Biol. Chem. 1997; 272: 29742-29751Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) (Fig. 1A). Co-immunoprecipitation experiments show that, like the wild type IκBβ, mutant IκBβΔ-(152–192) is also associated with p65 (Fig. 1C). Our results thus demonstrate that the insert of IκBβ plays a role in vivo in IκBβ·NF-κB complex cytoplasmic retention. We have also tested whether the insert of IκBβ is responsible for cytosolic retention of the IκBβ·NF-κB p50/p65 heterodimer complex (Fig. 1B). As in the case of the IκBβΔ-(152–192)·NF-κB p65 homodimer complex, we observe that the IκBβΔ-(152–192) protein in complex with NF-κB p50/p65 heterodimer can also enter the nucleus of resting cells. A likely explanation for this phenomenon is that when the insert of IκBβ is removed, the second NLS becomes free (or more loosely bound to IκBβ), enabling the complex to be actively imported into the nucleus. These results suggest that the p50 subunit in the p50/p65 heterodimer acts similarly to p65 subunit B in the NF-κB p65 homodimer, which possesses an NLS polypeptide that is primarily solvent-exposed. The p50 Subunit NLS Regulates Nuclear Translocation of IκBβ·NF-κB p50/p65 Heterodimer Complexes—To further test whether nuclear translocation of the IκBβΔ-(152–192)·NF-κB p50/p65 heterodimer complex is mediated by the free p50 NLS, we performed co-transfection experiments with a p50 subunit that lacks its NLS polypeptide (p50ΔNLS), p65, and IκBβΔ-(152–192) (Fig. 2). We observe that this complex is cytoplasmic in both the absence and presence of LMB, suggesting that only in the IκBβΔ-(152–192)·NF-κB p50/p65 heterodimer complex is the p50 NLS free, whereas it remains masked in the wild type IκBβ·NF-κB p50/p65 heterodimer complex. These results indicate that one NF-κB NLS and the IκBβ insert antagonize one another in directing subcellular localization of all IκBβ·NF-κB complexes. κB-Ras Regulates the p65 Subunit B NLS of the IκBβ·NF-κB p65 Homodimer Complex in Vitro and in Cells—The above experiments suggest that the IκBβ insert could mask the second (subunit B) NLS of IκBβ·NF-κB complexes in resting cells. Direct contact likely requires modification of one or both of these interacting elements. The small GTPase κB-Ras was recently shown to be involved in regulation of IκB degradation (36Fenwick C. Na S.Y. Voll R.E. Zhong H. Im S.Y. Lee J.W. Ghosh S. Science. 2000; 287: 869-873Crossref PubMed Scopus (88) Google Scholar). To determine whether κB-Ras might be involved in masking the NF-κB subunit B NLS, we have performed immunoprecipitation experiments with a monoclonal antibody directed against the p65 subunit NLS used to precipitate free p65, IκBβ·NF-κB p65 homodimer binary complexes, and κBRas·IκBβ·NF-κB p65 homodimer ternary complexes. In parallel experiments, IκBα·NF-κB p65 homodimer complexes were probed in the presence and absence of κB-Ras. The p65 from the immunoprecipitated complexes was then visualized by anti-p65 polyclonal antibody (Fig. 3A). We observe that both IκBβ·NF-κB p65 homodimer and IκBα·NF-κB p65 homodimer complexes can be precipitated by the anti-p65 NLS antibody (Fig. 3A, lanes 2 and 7, respectively). Moreover, we observe that the addition of κB-Ras inhibits the ability of this antibody to bind to IκBβ·NF-κB p65 homodimer complexes but not IκBα·NF-κB p65 homodimer (Fig. 3A, lanes 3 and 8). To determine whether the insert of IκBβ plays a role in NLS masking by κB-Ras, we performed similar immunoprecipitation experiments with IκBβΔ-(152–192)·NF-κB p65 homodimer complexes in the presence and absence of κBRas. We observe that the addition of κB-Ras fails to enhance NLS masking of IκBβΔ-(152–192)·NF-κB p65 homodimer complexes (Fig. 3A, lanes 4 and 5). To verify specificity of the anti-p65 NLS monoclonal antibody, we tested it against the IκBβ·NF-κB p50/p65 complex. The anti-p65 NLS antibody fails to recognize and precipitate p65 in these complexes. This results from the nearly complete masking of the p65 subunit A NLS polypeptide by IκBβ as observed in the IκBβ·NF-κB p65 complex crystal structure (Fig. 3A, lanes 9 and 10). To further investigate whether κB-Ras mediates blockade of the p65 NLS in cells, we next co-transfected 293 cells with HA-tagged p65 together with IκBα, IκBβ, or IκBβΔ-(152–192). We first confirmed that p65 associates with all three IκB proteins by co-immunoprecipitation using an antibody against the transcriptional activation domain of p65 (Fig. 3B). When the anti-p65 NLS antibody was used for immunoprecipitation, we observed that only IκBα and IκBβΔ-(152–192) were pulled down, but not wild type IκBβ. These experiments suggest that in IκBα·NF-κB p65 homodimer and IκBβΔ-(152–192) complexes, at least one p65 NLS is free, whereas both p65 subunit NLS polypeptides are blocked in the IκBβ·NF-κB p65 homodimer complex. We conclude that the IκBβ insert is required for masking of the NF-κB subunit B NLS and cytoplasmic retention of IκBβ·NF-κB complexes. κB-Ras Directly Interacts with the IκBβ Insert—To identify the role of the IκBβ insert in κB-Ras binding, we have tested the binding of co-transfected κB-Ras and IκBβ in cells. COS cells were co-transfected with plasmids expressing both wild type IκBβ and κB-Ras. We show that κB-Ras can be precipitated only when wild type IκBβ is present (Fig. 4). However, the association is not observed in cells expressing IκBβΔ-(152–192) and κB-Ras. Therefore, the IκBβ insert is required for association with κB-Ras. Taken together, these experiments suggest that the small GTPase κB-Ras may play an in vivo role in blocking the subunit B NLS of dimeric NF-κB by binding directly to the unique insert region of the IκBβ inhibitor protein. The IκBβ PEST Functions in Cytoplasmic Retention of IκBβ·NF-κB Complexes—Because κB-Ras was identified as an IκB-interacting protein in a yeast two-hybrid screen with the IκBβ C-terminal PEST used as the bait, it is important to evaluate the role of the IκBβ PEST in NF-κB cytoplasmic sequestration. To test whether phosphorylation of the IκBβ PEST plays any role in cytoplasmic retention of NF-κB, we mutated four phosphorylatable serines (Ser312, Ser313, Ser314, and Ser316) within this region to either alanine (PEST-Ala) or aspartic acid (PEST-Asp). Two serine residues in this region have been shown to be phosphorylated by casein kinase II, and phosphorylation at these sites is important for NF-κB binding in cells (39Chu Z.L. McKinsey T.A. Liu L. Qi X. Ballard D.W. Mol. Cell. Biol. 1996; 16: 5974-5984Crossref PubMed Google Scholar, 40McKinsey T.A. Chu Z.L. Ballard D.W. J. Biol. Chem. 1997; 272: 22377-22380Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). However, we have previously shown that phosphorylation of these serines or their conversion to phosphomimetic glutamic acid residues does not alter the stability of IκBβ·NF-κB binary complexes in vitro (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). If constitutive phosphorylation of the PEST is important for stable complex formation, then substitution of these residues to alanine could prevent cytoplasmic retention of IκBβ·NF-κB complexes. To test this possibility, we co-transfected HeLa cells with IκBβ-PEST-Ala and p65 and observed co-localization of both proteins in the cytoplasm. However, when these cells were treated with LMB followed by immunostaining, we observed that, in a large fraction of cells expressing both of these proteins, the complex localized to the nucleus (Fig. 5A). In contrast, when the cells were co-transfected with p65 and IκBβ-PEST-Asp, the complex was cytoplasmic with or without LMB treatment (Fig. 5B). This property of the IκBβ-PEST-Asp mutant complex is identical to that of the wild type complex. We conclude that IκBβ PEST phosphorylation is critical for cytoplasmic sequestration of IκBβ·NF-κB complexes. We further created a mutant of IκBβ in which the insert is deleted within the background of phosphomimetic IκBβ-PESTAsp sequence ((IκBβΔ-(152–192)-PEST-Asp). When HeLa cells were co-transfected with this mutant and wild type p65, we observed that the complex shuttled between the cytoplasm and nucleus (Fig. 5C). This profile is identical to that observed in the IκBβΔ-(152–192)·NF-κB p65 homodimer complex. It is not immediately clear to us what role a phosphorylated IκBβ PEST plays in sequestering the IκBβ·NF-κB complexes to the cytosol. It is possible that κB-Ras interacts with both the IκBβ insert and phosphorylated PEST. It is also possible that the interaction between the IκBβ PEST and κB-Ras is indirect, mediated by another bridging factor. IκBβ·NF-κB complexes are cytoplasmic in quiescent cells (30Huang T.T. Miyamoto S. Mol. Cell. Biol. 2001; 21: 4737-4747Crossref PubMed Scopus (89) Google Scholar, 31Tam W.F. Sen R.J. J. Biol. Chem. 2001; 276: 7701-7704Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This suggests that the NF-κB subunit B NLS can exhibit a binding mode, alternative to that observed in the IκBβ·NF-κB p65 homodimer complex crystal structure, in which it is completely masked (see Ref. 43Malek S. Huang D.-B. Huxford T. Ghosh S. Ghosh G. J. Biol. Chem. 2003; 278: 23094-23100Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We have tested this hypothesis derived from our structural analyses of IκB·NF-κB complexes by both in vitro and cell-based studies. Transient transfection and immunostaining experiments reveal that the unique and structurally disordered insert between the third and fourth ankyrin repeats of IκBβ functions to regulate nuclear import of the IκBβ·NF-κB p65 homodimer complex in resting cells. These experiments suggest that the insert functions as a nuclear export signal or blocks a nuclear localization sequence. No recognizable export sequence is observed within the IκBβ insert. Interestingly, IκBβΔ-(152–192)·NF-κB complexes shuttle between the nucleus and cytoplasm, despite the lack of a nuclear export sequence in IκBβ. We suggest that the partially exposed NLS of NF-κB subunit B and the export potential of the p65 activation domain drive the dynamic shuttling behavior of these complexes (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 35Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Crossref PubMed Scopus (86) Google Scholar). We propose the hypothesis that κB-Ras may act in concert with the IκBβ insert to sequester IκBβ·NF-κB complexes to the cytoplasm. Indeed, we show that the small GTPase κB-Ras is able to reduce access to the NF-κB p65 homodimer subunit B NLS in complexes between p65 and IκBβ. We further present evidence that binding of κB-Ras to IκBβ requires the insert within the ankyrin repeat domain of IκBβ. Because this insert is unique to IκBβ, these results serve to explain the specificity of κB-Ras for the IκBβ·NF-κB complex. There are several published observations which suggest that phosphorylation of the IκBβ PEST might play an important role in regulation of NF-κB by IκBβ (7Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 9Weil R. Whiteside S.T. Israël A. Immunobiology. 1997; 198: 14-23Crossref PubMed Scopus (21) Google Scholar, 22McKinsey T.A. Brockman J.A. Scherer D.C. Al-Murrani S.W. Green P.L. Ballard D.W. Mol. Cell. Biol. 1996; 16: 2083-2090Crossref PubMed Google Scholar). The most important of these is the identification of κB-Ras through the utilization of the IκBβ PEST sequence as bait in a yeast two-hybrid screen (36Fenwick C. Na S.Y. Voll R.E. Zhong H. Im S.Y. Lee J.W. Ghosh S. Science. 2000; 287: 869-873Crossref PubMed Scopus (88) Google Scholar). Also, it has been shown that PEST phosphorylation is important for stable complex formation with NF-κB (39Chu Z.L. McKinsey T.A. Liu L. Qi X. Ballard D.W. Mol. Cell. Biol. 1996; 16: 5974-5984Crossref PubMed Google Scholar). We show here that PEST phosphorylation of IκBβ functions in cytoplasmic retention of IκBβ·NF-κB complexes. Although we do not observe a clear need for PEST phosphorylation in the interaction between IκBβ and NF-κB or κB-Ras·IκBβ·NF-κB complex formation, our experiments do suggest that PEST phosphorylation cooperates with κB-Ras binding and the IκBβ insert in regulating cytosolic retention of IκBβ·NF-κB complexes. We propose that this cooperativity might be mediated through other cellular factors or through further post-translational modification elsewhere in the complex. We favor the first possibility in light of our recent findings that IκBβ is present in the cytoplasm of quiescent cells as large complexes, which include NF-κB and κB-Ras as well as other as-yet-unknown factors. 3Y. Chen, unpublished observation. Working together, the IκBβ insert and the PEST, one NF-κB NLS, and κB-Ras represent a molecular mechanism for switching between nuclear and cytoplasmic IκBβ·NF-κB complexes (Fig. 6). Our experiments help to explain the observation that hypophosphorylated IκBβ·NF-κB complexes can localize into the nucleus (7Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar). We suggest that the hypophosphorylated form of IκBβ is functionally equivalent to the IκBβ-PEST-Ala protein construct used in this study. Finally, our results serve to explain one long-standing puzzle. It has long been thought that IκBα binds NF-κB dimers with a significantly higher affinity than does IκBβ. This conclusion was drawn based on the respective abilities of IκBα and IκBβ to inhibit NF-κB DNA binding. We have shown previously that compared with IκBα, IκBβ binds NF-κB p50/p65 heterodimer and p65 homodimer with only slightly weaker affinity (32Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 41Malek S. Huxford T. Ghosh G. J. Biol. Chem. 1998; 273: 25427-25435Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 42Phelps C.B. Sengchanthalangsy L.L. Huxford T. Ghosh G. J. Biol. Chem. 2000; 275: 29840-29846Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In light of the IκBβ·NF-κB p65 homodimer complex crystal structure and the biochemical evidence surrounding κB-Ras binding to IκBβ·NF-κB complexes, we suggest that the in vivo stabilities of IκBβ·NF-κB complexes are nearly equivalent to those of IκBα·NF-κB complexes. This would explain why both complexes are present in almost all cells in almost equal amounts (5Whiteside S.T. Epinat J.C. Rice N.R. Israël A. EMBO J. 1997; 16: 1413-1426Crossref PubMed Scopus (337) Google Scholar). We thank H. Bantel and Roche Diagnostics for the generous gift of the anti-p65 NLS antibody, Dr. M. Yoshida for kindly supplying LMB, and T. Huxford, C. Phelps, and members of the G. Ghosh laboratory for commenting on the manuscript.