Horie R, Aizawa S, Nagai M, Ito K, Higashihara M, Ishida T, Inoue J, Watanabe T
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Int Immunol. 1998 Feb;10(2):203-10. doi: 10.1093/intimm/10.2.203.
About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFkappaB by CD30 was shown to be mediated through interaction of TNFR-associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFkappaB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFkappaB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFkappaB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFkappaB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFkappaB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFkappaB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFkappaB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C-terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFkappaB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.
肿瘤坏死因子受体（TNFR）超家族成员人、大鼠和小鼠的CD30，其C端区域约100个氨基酸残基是保守的，可分为三个保守性相对较高的亚结构域（D1、D2和D3）。已表明CD30对NFκB的激活是通过肿瘤坏死因子受体相关因子（TRAF）1、2和5与D2和D3亚结构域的相互作用介导的。然而，另一个保守亚结构域D1 的功能仍有待确定。D2和D3亚结构域的缺失消除了与TRAF2和5的相互作用，但不影响NFκB的激活。使用CD30的缺失和突变构建体进行的报告基因分析表明，D1亚结构域足以激活NFκB，无需与TRAF2或5相互作用，且每个亚结构域单独即可激活NFκB。电泳迁移率变动分析显示，在表达CD30或缺乏D2和D3亚结构域的缺失突变体的293细胞稳定转化体中，存在组成型和CD30诱导的NFκB激活。D1亚结构域C端19个氨基酸残基的缺失消除了NFκB的激活。将两个苏氨酸残基（氨基酸位置524和529）之一替换为丙氨酸，其中一个是D1亚结构域中的潜在磷酸化位点，也消除了NFκB的激活。TRAF2或5的TRAF结构域的过表达对由D1亚结构域介导的NFκB激活具有显性负效应，从而表明TRAF蛋白参与了信号传导。因此，CD30的C端100个氨基酸区域由三个独立的功能亚结构域组成，其中两个包含TRAF蛋白的结合位点。细胞质尾巴中的一个新结构域介导NFκB激活，无需TRAF2或5的直接相互作用。我们的观察结果表明，一种未知的TRAF蛋白参与了CD30的信号转导途径。
