Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex
Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex
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DOI：
10.1073/pnas.96.22.12436
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发表时间：
1999-10-26
影响因子：
11.1
通讯作者：
Pause, A
中科院分区：
文献类型：
--
作者：
Iwai, K;Yamanaka, K;Pause, A
von Hippel-Lindau病（VHL）肿瘤抑制基因产物（pVHL）突变见于显性遗传性VHL综合征患者和绝大多数散发的透明细胞肾癌。pVHL蛋白的功能尚未明确。pVHL与长蛋白B和长蛋白C （VBC）以及cullin (CUL)-2形成复合物。鉴于VBC-CUL-2与SKP1-CUL-1-F-box泛素连接酶在结构上的相似性，本研究检测了VBC-CUL-2的泛素连接酶活性。我们发现VBC-CUL-2具有泛素连接酶活性，我们鉴定出UbcH5a、b和c是VBC-CUL-2泛素连接酶的泛素结合酶，而不是CDC34。Rbx1/ROC1蛋白增强了VBC-CUL-2连接酶的活性，就像SKP1-CUL-1-F-box蛋白连接酶复合体一样。我们还发现pVHL与两个蛋白p100和p220结合，它们在泛素化实验中以相似的分子量作为两个主要条带迁移。此外，自然发生的pVHL错义突变，包括能够与长蛋白b -长蛋白C-CUL-2形成复合物的突变，不能与p100和pun结合，也不能表现出E3连接酶活性。这些结果表明pVHL可能是vbc - cu -2 E3连接酶的底物识别亚基。据我们所知，这也是人类肿瘤抑制蛋白直接参与泛素偶联系统导致底物蛋白靶向降解的第一个例子。
Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and pun and cannot exhibit the E3 ligase activity. These results suggest that pVHL might he the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.