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 dispensable 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 blocked 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.
Beauparlant
P
P
Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada.
Lin
R
R
Hiscott
J
J
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011480:Protease Inhibitors; D011506:Proteins; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D013752:Tetracycline