Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein
Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein
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DOI：
10.1074/jbc.m002740200
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Maxwell, PH
中科院分区：
文献类型：
--
作者：
Cockman, ME;Masson, N;Maxwell, PH
von Hippel-Lindau 肿瘤抑制蛋白 (pVHL) 已成为细胞对氧气可用性反应的关键因素，是缺氧诱导因子 1 (HIF) α 亚基的氧依赖性蛋白水解所必需的。VHL 突变会导致与血管生成失调和缺氧诱导因子上调相关的遗传性癌症综合征。 在这里，我们研究了这些过程背后的机制，并表明来自 VHL 缺陷的肾癌细胞的提取物在 HIF-α 泛素化活性方面存在缺陷，而外源性 pVHL 可以补充这一缺陷。在一系列测试的底物中，这种缺陷是 HIF-α 所特有的。此外，通过比较来自蛋白酶体抑制细胞和正常细胞的代谢标记的抗 pVHL 免疫沉淀物，HIF-α 亚基是唯一鉴定的 pVHL 相关蛋白酶体底物。对 pVHL/HIF-α 相互作用的分析确定了 HIF-α 分子内部反式激活结构域内的保守残基短序列足以被 pVHL 识别。相反，虽然全长 pVHL 和 p19 变体与 HIF-α 相互作用，但这种关联被进一步的 N 端和 C 端截短所消除。这种相互作用也被 pVHL β 结构域中肿瘤相关突变所破坏，相互作用的丧失与 HIF-α 泛素化和调节缺陷有关，定义了一种机制，通过这些突变产生促进血管生成的基因表达的组成性缺氧模式。研究结果表明，pVHL 通过充当识别因子来调节 HIF-α 蛋白水解。 泛素连接酶复合物的组成部分，并支持其 β 结构域与 HIF-α 亚基中的短识别序列相互作用的模型。
The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF), Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes, Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL, This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL, In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis, The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.