Pim D, Thomas M, Javier R, Gardiol D, Banks L
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Oncogene. 2000 Feb 10;19(6):719-25. doi: 10.1038/sj.onc.1203374.
The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We first show that the differences in the affinities of HPV-16 and of HPV-18 E6 proteins for binding DLG is reflected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to define regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Significant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most significantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway.
盘状大蛋白（DLG）肿瘤抑制蛋白是高危型人乳头瘤病毒（HPV）E6蛋白介导的泛素化降解的靶点。在本研究中，我们通过对E6进行突变分析来研究其发生机制。我们首先表明，HPV - 16和HPV - 18 E6蛋白与DLG结合亲和力的差异反映在它们各自将DLG作为降解靶点的能力上。对HPV - 18 E6的突变分析使我们能够确定该蛋白羧基末端一半区域内对于E6指导DLG降解能力至关重要的区域。通过降解p53的能力测定，E6氨基末端部分丧失结合E6 - AP泛素连接酶能力的突变体，仍然保留降解DLG的能力。使用缺乏E6 - AP的小麦胚芽提取物也能实现显著水平的DLG降解。最后，我们表明将DLG结合结构域转移到低危型HPV - 6 E6上赋予了该蛋白DLG结合活性，并且最重要的是，使HPV - 6 E6能够将DLG作为降解靶点。这些结果表明，E6介导的DLG降解不涉及E6 - AP泛素连接酶，此外，还表明高危型和低危型HPV E6蛋白很可能在泛素途径中共享一个共同的细胞中介物。
