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

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