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 engi neered to inducibly express wild type or mutated human I kappa B alpha proteins under the control of the tetracycline responsive promoter. D eletion 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), i ncluding 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 k appa 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 kapp a B alpha Delta 1 and I kappa B alpha Delta 2 mutants were resistant t o inducer-mediated degradation, Analysis of I kappa B alpha deletions in the presence of protein synthesis inhibitors revealed that, indepen dently of stimulation, I kappa B alpha Delta 1 and I kappa B alpha Del ta 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 De lta 2 with p65 was dramatically decreased in vivo as measured by co-im munoprecipitation. Interestingly, protease inhibitors which block indu cer-mediated degradation of I kappa B alpha also stabilized the turnov er of I kappa B alpha Delta 1 and I kappa B alpha Delta 2. Based on th ese studies, we propose that in the absence of stimulation, the C-term inal domain between aa 269 and 287 may Play a role to protect I kappa B alpha from a constitutive protease activity.