Efficient nuclear export of p65-IκBα complexes requires 14-3-3 proteins

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Abstract
IκB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFκB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFκB signaling pathway by physically interacting with p65 and IκBα 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 IκBα in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IκBα results in a predominantly nuclear distribution of both proteins. TNFα treatment promotes recruitment of 14-3-3 and IκBα to NFκB-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-IκBα complexes and the constitutive association of p65 with the chromatin. In this situation, NFκB-dependent genes become unresponsive to TNFα stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IκBα-p65 complexes and are required for the appropriate regulation of NFκB signaling.