The transcriptional activity of NF-kappa B is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CPB/p300 associates with NF-kappa B p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-kappa B by modulating its interaction with CBP/p300.

Homodimers of the NF-kappa B p50 subunit are transcriptionally repressive in cells, whereas they can promote transcription in vitro, suggesting that their endogenous effects are mediated by association with other factors. We now demonstrate that transcriptionally inactive nuclear NF-kappaB in resting cells consists of homodimers of either p65 or p50 complexed with the histone deacetylase HDAC-1. Only the p50-HDAC-1 complexes bind to DNA and suppress NF-kappa B-dependent gene expression in unstimulated cells. Appropriate stimulation causes nuclear localization of NF-kappa B complexes containing phosphorylated p65 that associates with CBP and displaces the p50-HDAC-1 complexes. Our results demonstrate that phosphorylation of p65 determines whether it associates with either CBP or HDAC-1, ensuring that only p65 entering the nucleus from cytoplasmic NF-kappa B:Ikappa B complexes can activate transcription.

Stimulation of cells with inducers of NF-kappaB such as LPS and IL-1 leads to the degradation of IkappaB-alpha and IkappaB-beta proteins and translocation of NF-kappaB to the nucleus. We now demonstrate that, besides the physical partitioning of inactive NF-kappaB to the cytosol, the transcriptional activity of NF-kappaB is regulated through phosphorylation of NF-kappaB p65 by protein kinase A (PKA). The catalytic subunit of PKA (PKAc) is maintained in an inactive state through association with IkappaB-alpha or IkappaB-beta in an NF-kappaB-IkappaB-PKAc complex. Signals that cause the degradation of IkappaB result in activation of PKAc in a cAMP-independent manner and the subsequent phosphorylation of p65. Therefore, this pathway represents a novel mechanism for the cAMP-independent activation of PKA and the regulation of NF-kappaB activity.