Yaron A, Hatzubai A, Davis M, Lavon I, Amit S, Manning A M, Andersen J S, Mann M, Mercurio F, Ben-Neriah Y
The Lautenberg Center for Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Nature. 1998 Dec 10;396(6711):590-4. doi: 10.1038/25159.
NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
核因子-κB是一种普遍存在的、可诱导的转录因子，参与免疫、炎症、应激和发育过程，在未受刺激的细胞胞质中，它通过抑制分子IκB以潜伏形式保留。其激活是信号转导级联反应的一个范例，该级联反应整合了一种可诱导激酶和泛素-蛋白酶体系统以消除抑制性调节因子。在此，我们分离出了pIκBα泛素连接酶（pIκBα-E3），它将泛素（一种标记其他蛋白质以便被蛋白酶体系统降解的小蛋白质）连接到磷酸化的核因子-κB抑制剂pIκBα上。利用其对pIκBα的高亲和力，我们通过单步免疫亲和纯化从HeLa细胞中分离出这种连接酶。使用纳米电喷雾质谱法，我们鉴定出该连接酶识别pIκBα降解基序的特定成分是一种属于最近区分出的β-TrCP/Slimb蛋白家族的F-box/ WD结构域蛋白。我们将该成分命名为E3RSIκB（pIκBα-E3受体亚基），它特异性结合pIκBα，并在另外两种泛素系统酶E1和UBC5C（众多已知E2酶之一）存在的情况下促进其体外泛素化。E3RS（IκB）的F-box缺失突变体紧密结合pIκBα但不支持其泛素化，在体内作为一种显性负性分子起作用，抑制pIκBα的降解，从而抑制核因子-κB的激活。E3RS（IκB）代表一类受体蛋白家族，它们是一类泛素连接酶的核心成分。当这些受体成分识别其特定配体（一种基于磷酸化的保守序列基序）时，它们将含有该基序的调节蛋白靶向蛋白酶体降解。
