A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes.
Appropriate subcellular localization is crucial for regulation of NF-kappaB function. Herein, we show that latent NF-kappaB complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-kappaB/IkappaBalpha complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45-54 of IkappaBalpha that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-kappaB nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-kappaB/IkappaBalpha complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-kappaB activation without affecting the subcellular localization of upstream kinases IKKbeta and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-kappaB activation by extracellular signals.
Huang
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TT
Program in Molecular and Cellular Pharmacology, Department of Pharmacology, University of Wisconsin, K4/554 Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792, USA.
Kudo
N
N
Yoshida
M
M
Miyamoto
S
S
D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002352:Carrier Proteins / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D028884:Karyopherins; D046911:Macromolecular Substances; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; D020672:I-kappa B Proteins; D028884:Karyopherins; D046911:Macromolecular Substances; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000097606:Exportin 1 Protein; C000606204:NFKBIA protein, human; C038753:leptomycin B