Beauparlant P, Lin R, Hiscott J
Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1996 May 3;271(18):10690-6. doi: 10.1074/jbc.271.18.10690.
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.
在本研究中，我们在经基因工程改造、能在四环素反应性启动子控制下诱导表达野生型或突变型人IκBα蛋白的NIH 3T3细胞中，研究了IκBα C末端在NF-κB/IκBα调节中的作用。缺失研究表明，C末端最后30个氨基酸（第288至317个氨基酸，在IκBαδ3中缺失），包括大部分PEST结构域，对于IκBα功能而言是可有可无的。然而，从第261至317个氨基酸或第269至317个氨基酸的缺失（分别为IκBαδ1和IκBαδ2），在体外缺乏解离NF-κB/DNA复合物的能力，并且无法抑制NF-κB依赖性转录。此外，IκBαδ1和IκBαδ2突变体对诱导剂介导的降解具有抗性。在存在蛋白质合成抑制剂的情况下对IκBα缺失进行分析发现，与刺激无关，IκBαδ1和IκBαδ2的半衰期比野生型IκBα短四倍，并且通过免疫共沉淀测定，IκBαδ1和IκBαδ2与p65在体内的相互作用显著降低。有趣的是，阻断诱导剂介导的IκBα降解的蛋白酶抑制剂也稳定了IκBαδ1和IκBαδ2的周转。基于这些研究，我们提出，在没有刺激的情况下，第269至287个氨基酸之间的C末端结构域可能起到保护IκBα免受组成型蛋白酶活性影响的作用。
