Liu Xuefeng, Yuan Hang, Fu Baojin, Disbrow Gary L, Apolinario Tania, Tomaic Vjekoslav, Kelley Melissa L, Baker Carl C, Huibregtse Jon, Schlegel Richard
Department of Pathology and Oncology, Georgetown University Medical School, 3900 Reservoir Rd. NW, Washington, DC 20057, USA.
J Biol Chem. 2005 Mar 18;280(11):10807-16. doi: 10.1074/jbc.M410343200. Epub 2005 Jan 17.
Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.
大多数人类癌细胞表现出端粒酶活性增加，这似乎对细胞的持续增殖和肿瘤形成至关重要。与恶性肿瘤相关的人乳头瘤病毒的E6蛋白至少部分地通过端粒酶催化亚基hTERT的转录激活来增加原代人角质形成细胞中的细胞端粒酶。在本研究中，我们调查了E6AP（一种以结合并介导E6癌蛋白的某些活性而闻名的泛素连接酶）是否参与hTERT启动子的反式激活。我们的结果表明，无法结合E6AP的E6突变体在增加端粒酶活性和反式激活hTERT启动子方面也存在缺陷。更重要的是，E6AP基因敲除小鼠细胞和小干扰RNA技术表明，E6AP在小鼠和人类细胞中都是hTERT启动子反式激活所必需的。在没有伴侣蛋白的情况下，E6和E6AP都不会与hTERT启动子结合或激活该启动子。对于所有具有反式激活能力的E6蛋白，hTERT启动子的诱导依赖于核心启动子中的E盒元件。因此，似乎E6介导的hTERT启动子激活需要E6 - E6AP复合物与hTERT启动子结合，并且激活依赖于启动子中的Myc结合位点。在E6介导的转录激活过程中，一种细胞泛素连接酶被招募到hTERT启动子，这表明启动子相关调节蛋白（包括Myc蛋白）的局部泛素化（以及潜在的降解）发挥了作用。
