Rottjakob E M, Sachdev S, Leanna C A, McKinsey T A, Hannink M
Department of Biochemistry, University of Missouri, Columbia 65212, USA.
J Virol. 1996 May;70(5):3176-88. doi: 10.1128/JVI.70.5.3176-3188.1996.
Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.
c-Rel与κB抑制因子α（IkappaB-α）蛋白的结合调控细胞定位和DNA结合。禽源c-Rel的致癌病毒对应物v-Rel逃避禽源IkappaB-α蛋白p40调控的能力，促成了v-Rel介导的肿瘤发生。利用酵母双杂交系统在体内剖析Rel:IkappaB-α的相互作用。我们发现，c-Rel和v-Rel中不同的结构域与p40的结合是必需的。此外，虽然p40的锚蛋白重复结构域足以与c-Rel结合，但与v-Rel结合则需要锚蛋白重复结构域和PEST结构域。鉴定出c-Rel和v-Rel之间两个主要负责v-Rel与p40的PEST依赖性结合的氨基酸差异。相同的氨基酸主要负责p40对v-Rel的PEST依赖性细胞质滞留。c-Rel中足以赋予突变型c-Rel蛋白与p40的PEST依赖性结合的突变的存在，并未增加c-Rel的弱致癌性。然而，将这两个源自c-Rel的氨基酸引入v-Rel显著降低了v-Rel的致癌性。删除c-Rel或v-Rel的核定位信号（NLS）并未消除与p40的结合，但确实赋予了c-Rel与p40的PEST依赖性结合。令人惊讶的是，删除v-Rel中的核定位信号并不影响v-Rel的致癌性。对几种突变型c-Rel和v-Rel蛋白的分析表明，Rel蛋白与p40的结合对于细胞质滞留是必要的，但并不充分。这些结果与p40通过相同机制调节v-Rel和c-Rel的细胞定位这一假设不一致。相反，这些结果支持p40通过不同机制调节v-Rel和c-Rel的细胞定位这一假设。
