The role of the C-terminal domain of I kappa B alpha in protein degradation and stabilization

In the present study, the role of the I kappa B alpha C terminus in NF-kappa B/I kappa B alpha regulation was examined in NIH 3T3 cells engineered to inducibly express wild type or mutated human I kappa B alpha 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 I kappa B alpha Delta 3), including most of the PEST domain, were dispensible for I kappa B alpha function. However, deletions from aa 261 to 317 or aa 269 to 317 (I kappa B alpha Delta 1 and I kappa B alpha Delta 2 respectively), lacked the ability to dissociate NF-kappa B/DNA complexes in vitro and were unable to inhibit NF-kappa B dependent transcription. Moreover, I kappa B alpha Delta 1 and I kappa B alpha Delta 2 mutants were resistant to inducer-mediated degradation, Analysis of I kappa B alpha deletions in the presence of protein synthesis inhibitors revealed that, independently of stimulation, I kappa B alpha Delta 1 and I kappa B alpha Delta 2 had a half-life four times shorter than wild type I kappa B alpha and the interaction of I kappa B alpha Delta 1 and I kappa B alpha Delta 2 with p65 was dramatically decreased in vivo as measured by co-immunoprecipitation. Interestingly, protease inhibitors which block inducer-mediated degradation of I kappa B alpha also stabilized the turnover of I kappa B alpha Delta 1 and I kappa B alpha Delta 2. 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 I kappa B alpha from a constitutive protease activity.