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.

Extracellular stimuli that activate the transcription factor NF-B cause rapid phosphorylation of the IB␣ inhibitor, which retains NF-B in the cytoplasm of nonstimulated cells. Phosphorylation of IB␣ is followed by its rapid degradation, the inhibition of which prevents NF-B activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IB␣. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IB␣ degradation. Substitutions of the homologous sites in IB␤, serines 19 and 23, also prevent inducible IB␤ degradation. We suggest that activation of a single IB kinase or closely related IB kinases is the first critical step in NF-B activation. Once phosphorylated, IB is ubiquitinated. Unlike wild-type IB␣, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IB␣ without affecting its phosphorylation, polyubiquitination is required for inducible IB degradation.The transcription factor NF-B is composed of heterodimeric and homodimeric complexes of the Rel proteins p50 (NF-B1), p52 (NF-B2), p65 (RelA), RelB, and c-Rel (reviewed in references 7, 22, 33, and 42). Originally described as a constitutively active nuclear factor in B lymphocytes (41), NF-B is cytoplasmic in most other cell types, but following exposure to a variety of extracellular stimuli, it translocates to the nucleus and activates transcription of specific target genes (reviewed in references 4, 23, and 42). The cytoplasmic retention of NF-B in nonstimulated cells is mediated by a family of inhibitor proteins, the IBs (reviewed in references 5 and 20). As specific molecular reagents were first available for IB␣, more is known about its regulation than about other IBs. Exposure of cells to NF-B-activating stimuli, including tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), lipopolysaccharide (LPS), 12-O-tetradecanoylphorbol-13-acetate (TPA), and UV irradiation, results in rapid IB␣ degradation (6,11,14,15,27,34). Recently some of these stimuli were also shown to trigger degradation of IB␤, albeit with kinetics different from that for IB␣ degradation (44). In addition, inhibitors of IB␣ degradation, most notably peptide aldehydes that also inhibit the multifunctional protease (26S proteasome), were found to prevent NF-B activation (2,16,17,32,38,39,46). It was therefore suggested that degradation of IB by the proteasome is an essential early step in the NF-B activation pathway (reviewed in references 13 and 43).Degradation of IB␣ is preceded by its phosphorylation. Although phosphorylation of IB␣ does not cause its dissociation from NF-B, it appears that the phosphorylated form may be preferentially degraded (2,16,17,32,38,46). Phosphopeptide mapping suggested that inducible IB␣ phosphorylation occurs on a small number of site...

Stimulation with inducers that cause persistent activation of NF-B results in the degradation of the NF-B inhibitors, IB␣ and IB␤. Despite the rapid resynthesis and accumulation of IB␣, NF-B remains induced under these conditions. We now report that IB␤ is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated IB␤ forms a stable complex with NF-B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-B, and the IB␤-NF-B complex enters the nucleus. It appears therefore that during prolonged stimulation, IB␤ functions as a chaperone for NF-B by protecting it from IB␣ and allowing it to be transported to the nucleus.The transcription factor NF-B is a ubiquitously expressed transcription factor that plays an important role in the inducible expression of a large number of cellular and viral genes (3,15,24). In the majority of cells, NF-B exists in an inactive form in the cytoplasm by being bound to the inhibitory protein IB␣ or IB␤ (2,25,26). Treatment of cells with various inducers results in the degradation of IB proteins, thus releasing the bound NF-B, which translocates to the nucleus and upregulates gene expression (4,8,9,13,(21)(22)(23)25). IB␣ is degraded by all of the known inducers of NF-B, whereas IB␤ is degraded only when cells are stimulated with inducers such as lipopolysaccharide (LPS) and interleukin-1 (IL-1) which cause persistent activation of NF-B. In either case, active NF-B causes an upregulation of IB␣ mRNA levels as a result of the presence of NF-B sites in the IB␣ promoter (10, 16). The newly synthesized IB␣ helps to terminate the NF-B response by resequestering NF-B. However, in persistent activation by LPS or IL-1, some of the NF-B appears to be insensitive to newly made IB␣ (25). It is unknown, however, how this pool of NF-B escapes inhibition by the newly made IB␣ and why only inducers that affect IB␤ are able to cause persistent activation.We report here that following degradation of the initial pool of IB-␤ in response to inducers such as LPS or IL-1, newly synthesized IB␤ accumulates as an unphosphorylated protein that forms a stable complex with a portion of NF-B and prevents it from binding to newly synthesized IB␣. The IB␤ in its unphosphorylated state differs from basally phosphorylated IB␤, as it is unable to mask the nuclear localization signal (NLS) and the DNA binding domain of NF-B. Therefore, the NF-B bound to unphosphorylated IB␤ can enter the nucleus and bind to DNA. Hence, during persistent activation of NF-B, the newly synthesized, unphosphorylated IB␤ plays a chaperone-like role, by binding to a portion of newly synthesized NF-B and allowing it to be transported to the nucleus. MATERIALS AND METHODSImmunoblot analysis. Immunoblot analysis was generally carried out with 25 to 30 g of extract. Following fractionation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the proteins were electrophoretically transferred to polyvinylidene difluoride m...