Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.
IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.
Aguilera
Cristina
C
Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Gran Via km 2.7, Hospitalet, Barcelona 08907, Spain.
Fernández-Majada
Vanessa
V
Inglés-Esteve
Julia
J
Rodilla
Verónica
V
Bigas
Anna
A
Espinosa
Lluís
L
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes; D000072000:NF-KappaB Inhibitor alpha; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D048948:14-3-3 Proteins; D002843:Chromatin; D020672:I-kappa B Proteins; D046912:Multiprotein Complexes; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha