Thatte Jayashree, Banks Lawrence
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
J Virol. 2017 Oct 27;91(22). doi: 10.1128/JVI.01390-17. Print 2017 Nov 15.
The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution. Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a redirection of E6AP activity toward itself. Furthermore, E6-mediated regulation of the subcellular distribution of phospho-E6AP appears to be dependent, in part, upon the 14-3-3 family of proteins.
人乳头瘤病毒（HPV）E6癌蛋白募集细胞泛素连接酶E6AP/UBE3A，以将细胞底物靶向蛋白酶体介导的降解，这种活性的一个结果是E6对E6AP自身泛素化和降解的刺激。最近的研究在E6AP的T485位点发现了一个与自闭症相关的突变，该位点被鉴定为蛋白激酶A磷酸化位点，并且可以直接调节E6AP泛素连接酶活性。在本研究中，我们分析了T485介导的E6AP调节如何影响E6对其一些已知底物的靶向作用。我们发现T485的调节对E6指导p53或Dlg降解的能力没有影响。此外，T485调节对HPV-16或HPV-31 E6诱导的E6AP自降解没有影响，但确实影响HPV-18 E6诱导的E6AP自降解。在源自宫颈癌的细胞中，我们发现细胞核中磷酸化和非磷酸化的E6AP水平都很低。然而，E6的缺失导致磷酸化E6AP在细胞质中大量积累，而非磷酸化E6AP主要在细胞核中积累。有趣的是，T485位点的E6AP磷酸化赋予其与14-3-3蛋白的结合能力，并且这种相互作用似乎在一定程度上对E6将磷酸化E6AP募集到细胞核中的能力很重要。这些结果表明，HPV E6在其酶活性和亚细胞分布方面都超越了E6AP的正常磷酸化调节。最近的报道证明了E6AP磷酸化调节对其正常酶活性的重要性。在这里，我们表明HPV E6能够超越这种调节，并且无论E6AP的磷酸化状态如何，都可以促进p53和Dlg的降解。此外，E6与E6AP的相互作用也显著改变了E6AP如何进行自降解，这表明这不是对已存在活性的简单刺激，而是E6AP活性对自身的重新定向。此外，E6介导的磷酸化E6AP亚细胞分布调节似乎部分依赖于14-3-3蛋白家族。
