IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells.
NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.
Malek
S
S
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0359, USA.
Chen
Y
Y
Huxford
T
T
Ghosh
G
G
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry*; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D019913:Nuclear Localization Signals; D000072000:NF-KappaB Inhibitor alpha; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; D004247:DNA; D011392:Proline