Suzuki H, Chiba T, Kobayashi M, Takeuchi M, Furuichi K, Tanaka K
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Ibaraki, Tsukuba-shi, 305-8585, Japan.
Biochem Biophys Res Commun. 1999 Mar 5;256(1):121-6. doi: 10.1006/bbrc.1999.0296.
Activation of the transcriptional factor NF-kappaB is triggered by signal-dependent degradation of its inhibitor protein IkappaB through the ubiquitin (Ub)-proteasome pathway. We found here that a phosphorylated IkappaBalpha immunoprecipitated (IP-pIkappaBalpha) from the crude extract of HeLa cells which had been treated with tumor necrosis factor-alpha (TNFalpha) caused a dramatic ubiquitination of itself, termed autoubiquitination, when incubated with ATP, Ub, and E1-activating and E2-conjugating enzymes. IP-pIkappaBalpha also catalyzed ubiquitination of an in vitro synthesized 35S-IkappaBalpha previously phosphorylated by IkappaB-kinase (IKK) which is referred to as transubiquitination. No appreciable activity of auto- and transubiquitination was observed in an unphosphorylated IP-IkappaBalpha. Moreover, the putative IkappaBalpha-Ub ligase (IkappaBalpha-E3) present in HeLa cell cytosol associated in vitro with an IKK-phosphorylated recombinant IkappaBalpha, a process independent of NF-kappaB binding to IkappaBalpha or TNFalpha stimulation. Replacement of the two Ser residues at positions 32 and 36 corresponding to IKK phosphorylation sites by Ala resulted in almost complete prevention of binding of an IkappaBalpha-E3 to IkappaBalpha. These results indicate that phosphorylation of IkappaBalpha is necessary and sufficient for recruitment of this IkappaBalpha-E3 to associate with IkappaBalpha.
转录因子NF-κB的激活是由其抑制蛋白IκB通过泛素（Ub）-蛋白酶体途径进行信号依赖性降解所触发的。我们在此发现，从用肿瘤坏死因子-α（TNFα）处理过的HeLa细胞粗提物中免疫沉淀得到的磷酸化IκBα（IP-pIκBα），在与ATP、Ub以及E1激活酶和E2缀合酶一起孵育时，会引发自身的显著泛素化，即自泛素化。IP-pIκBα还催化了先前由IκB激酶（IKK）磷酸化的体外合成的35S-IκBα的泛素化，这被称为转泛素化。在未磷酸化的IP-IκBα中未观察到明显的自泛素化和转泛素化活性。此外，HeLa细胞胞质溶胶中存在的假定的IκBα-Ub连接酶（IκBα-E3）在体外与IKK磷酸化的重组IκBα结合，这一过程独立于NF-κB与IκBα的结合或TNFα刺激。将对应于IKK磷酸化位点的第32和36位的两个丝氨酸残基替换为丙氨酸，几乎完全阻止了IκBα-E3与IκBα的结合。这些结果表明，IκBα的磷酸化对于募集这种IκBα-E3与IκBα结合是必要且充分的。
