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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...

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