Transactivation Domain 2 (TA) of p65 NF-B

Abstract

The p65 subunit of the inducible transcription factor NF-κB contains at least two strong transactivation domains (TADs) within its C terminus. The first domain, TA¹, is contained within the last 30 amino acids of p65, whereas TA² comprises the adjacent 90 amino acids. In this study, squelching experiments revealed that both TADs of p65, as well as the related subunit c-Rel, compete for the same cofactor(s) mediating transactivation. Both TADs of p65 share a common sequence motif, which is evolutionarily conserved and displays a remarkable degree of spatial organization when aligned on an α-helical surface. The functional importance of the common sequence motif was confirmed by deletion analysis of TA². Within the conserved sequence motif, a 7-amino-acid repeat was noted. Idealized heptad repeats fused to the DNA binding domain of Gal4 were transcriptionally active, but only as multimers. Phosphorylation and transcriptional activity of a defined region within the TA² domain was found to be stimulated by phorbol ester treatment of cells. In contrast, TA¹ was constitutively phosphorylated, and its activity did not significantly respond to phorbol ester stimulation. The stimulatory effect of phorbol ester on transcription of the TA² domain was completely blocked by the protein kinase C inhibitor. These data suggest that protein kinase C has a dual effect on NF-κB activity. It not only causes removal of IκB-α from cytoplasmic NF-κB but also augments the transactivation potential of activated nuclear NF-κB.