An N-terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IkappaBalpha.
The potent transcriptional activities of Rel/NF-kappaB proteins are regulated in the cytoplasm and nucleus by the inhibitor, IkappaBalpha. The mechanism, by which IkappaBalpha can either sequester NF-kappaB in the cytoplasm or act as a nuclear post-induction repressor of NF-kappaB, is uncertain. We find that IkappaBalpha shuttles continuously between the nucleus and cytoplasm. This shuttling requires a previously unidentified CRM1-dependent nuclear export signal (NES) located within the N-terminal domain of IkappaBalpha at amino acids 45-55. Deletion or mutation of the N-terminal NES results in nuclear localization of IkappaBalpha. NF-kappaB (p65) association with IkappaBalpha affects steady-state localization but does not inhibit its shuttling. Endogenous complexes of IkappaBalpha-NF-kappaB shuttle and will accumulate in the nucleus when CRM1 export is blocked. We find TNFalpha can activate the nuclear IkappaBalpha-NF-kappaB complexes by the classical mechanism of proteasome-mediated degradation of IkappaBalpha. These studies reveal a more dynamic nucleocytoplasmic distribution for IkappaBalpha and NF-kappaB suggesting previously unknown strategies for regulating this ubiquitous family of transcription activators.
Johnson
C
C
Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Van Antwerp
D
D
Hope
T J
TJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D051996:Transcription Factor RelA; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase