Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant.
Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378.
Holly E
HE
Dembinski
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378.
Kevin
K
Wismer
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095.|Institute for Quantitative Computational Biosciences, University of California, Los Angeles, CA 90095.
Jesse D
JD
Vargas
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095.|Institute for Quantitative Computational Biosciences, University of California, Los Angeles, CA 90095.
Gajendra W
GW
Suryawanshi
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378.
Nadja
N
Kern
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037.
Gerard
G
Kroon
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037.
H Jane
0000-0001-6855-3398
HJ
Dyson
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095.|Institute for Quantitative Computational Biosciences, University of California, Los Angeles, CA 90095.
Alexander
A
Hoffmann
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378; ekomives@ucsd.edu.
Elizabeth A
EA
Komives
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D055786:Gene Knockout Techniques; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics; D051379:Mice; D062105:Molecular Docking Simulation; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D013312:Stress, Physiological / Q000502:physiology; D051996:Transcription Factor RelA / Q000235:genetics; D015533:Transcriptional Activation
C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C491366:Nfkbie protein, mouse; D011518:Proto-Oncogene Proteins; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA
binding kinetics; hydrogen–deuterium exchange; intrinsically disordered proteins; nuclear export; transcription factor