The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells
The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells
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DOI：
10.1074/jbc.273.11.6439
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发表时间：
1998-03-13
影响因子：
4.8
通讯作者：
Howley, PM
中科院分区：
文献类型：
--
作者：
Talis, AL;Huibregtse, JM;Howley, PM
由致癌人乳头瘤病毒 (HPV) 编码的 E6 蛋白以 p53 为目标，进行泛素依赖性蛋白水解。 E6 介导的 p53 降解需要 100 kDa 细胞蛋白 E6 相关蛋白 (E6AP)。 E6AP和E6共同提供E3-泛素蛋白连接酶活性，将泛素转移到p53。体外研究表明，E6AP可以与泛素形成高能硫醇酯键，并且在E6存在的情况下，将泛素转移到p53。在这项研究中，我们探讨了 E6AP 在体内 p53 降解中的作用。 HPV18 阳性并表达 E6 的 HeLa 细胞中轻度型 E6AP 过度表达，导致 p53 稳态水平降低和 p53 半衰期缩短。鉴定出 E6AP 蛋白的突变形式，它们不能催化参与 p53 的 E6 依赖性泛素化，并且以显性失活方式发挥作用，因为它们在体外抑制野生型 E6AP 对 E6 介导的 p53 泛素化。这些显性失活 (dn) 突变体之一的瞬时转染导致 HeLa 细胞中 p53 体内稳态水平和半衰期的增加。与这一观察结果一致，dn E6AP 的过度表达导致细胞周期谱发生显着的 G(1) 转变。相比之下，dn E6AP 对 U2OS 细胞（一种含有野生型 p53 的 HPV 阴性细胞系）中的 p53 水平没有影响。这些研究为E6AP参与E6介导的p53体内降解提供了证据，并且还表明在E6缺失的情况下E6AP可能不参与p53泛素化的调节。
The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53, In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of mild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G(1) shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6.