Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IκBα
Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IκBα
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DOI：
10.1074/jbc.274.21.14823
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发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Ciechanover, A
中科院分区：
文献类型：
--
作者：
Gonen, H;Bercovich, B;Ciechanover, A
转录因子NF-κ B活化的最后一步是信号诱导的、泛素和蛋白酶体介导的抑制剂I κ B α降解。虽然参与活化和降解途径的大多数组分已经被鉴定，但泛素载体蛋白（E2）仍然难以捉摸。在这里，我们表明，两个高度同源的UBCH 5家族成员，UBCH 5 b和UBCH 5c，和CDC 34/UBC 3，酵母Cdc 34/Ubc 3的哺乳动物同源物，是E2酶参与的过程。它们在体外催化的偶联反应是特异性的，因为它们不识别I κ B α的S32 A、S36 A突变体，这些突变体不能在细胞外信号后被磷酸化和偶联。此外，该反应被跨越抑制剂的泛素连接酶识别结构域的双磷酸化肽特异性抑制。不能结合泛素的酶的Cys-至-Ala突变体种类在体内抑制肿瘤坏死因子α诱导的抑制剂降解。毫不奇怪，它们在无细胞系统中也有类似的效果。虽然很明显E2酶对I κ B α不是完全特异性的，但它们也不参与大量细胞蛋白质的缀合和降解，因此表现出一定程度的特异性，这可能是通过它们与泛素-蛋白质连接酶的确定子集的结合介导的。两种不同的E2参与I κ B α结合的机制目前尚不清楚。不同的接合机制可能在不同的生理条件下或在不同的细胞中运行。
The last step in the activation of the transcription factor NF-kappa B is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor I kappa B alpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of I kappa B alpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor a-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to I kappa B alpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in I kappa B alpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.