In the present study, the role of the IB C terminus in NF-B/IB regulation was examined in NIH 3T3 cells engineered to inducibly express wild type or mutated human IB proteins under the control of the tetracycline responsive promoter. Deletion studies demonstrated that the last C-terminal 30 amino acids (amino acids (aa) 288 to aa 317, deleted in IB3), including most of the PEST domain, were dispensible for IB function. However, deletions from aa 261 to 317 or aa 269 to 317 (IB1 and IB2 respectively), lacked the ability to dissociate NF-B/DNA complexes in vitro and were unable to inhibit NF-B dependent transcription. Moreover, IB1 and IB2 mutants were resistant to inducer-mediated degradation. Analysis of IB deletions in the presence of protein synthesis inhibitors revealed that, independently of stimulation, IB1 and IB2 had a half-life four times shorter than wild type IB and the interaction of IB1 and IB2 with p65 was dramatically decreased in vivo as measured by co-immunoprecipitation. Interestingly, protease inhibitors which block inducer-mediated degradation of IB also stabilized the turnover of IB1 and IB2. Based on these studies, we propose that in the absence of stimulation, the C-terminal domain between aa 269 and 287 may play a role to protect IB from a constitutive protease activity.

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