The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells
The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells
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DOI：
10.1371/journal.ppat.1007769
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发表时间：
2019-05-01
期刊：
影响因子：
6.7
通讯作者：
Banks, Lawrence
中科院分区：
文献类型：
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作者：
Basukala, Om;Mittal, Suruchi;Banks, Lawrence
人乳头瘤病毒E7癌蛋白在恶性肿瘤的发展和维持中起着重要作用，它通过靶向许多关键的细胞控制途径来实现。E7促进细胞转化能力的一个重要因素是在蛋白质的CR2结构域内存在酪蛋白激酶II磷酸受体位点。磷酸化被认为增强E7与许多不同细胞靶蛋白的相互作用，从而增加E7增强细胞增殖和诱导恶性肿瘤的能力。然而，一旦肿瘤细胞完全转化，关于E7中的这个位点有多重要的信息很少。在这项研究中，我们对C4-1宫颈肿瘤衍生细胞中的HPV-18 E7 CKII识别位点进行了基因组编辑。我们首先表明HPV 18 E7 S32/S34突变为A32/A34消除了E7的CKII磷酸化，随后我们分离了在E7中含有这些突变的C4-1克隆。细胞继续增殖，但比野生型细胞生长得更慢，达到更低的饱和密度，并且也更容易受到低营养条件的影响。这些细胞在基质胶侵袭测定中严重缺陷，部分原因是基质金属蛋白酶（MMP）下调。从机制上讲，我们发现E7的磷酸化在E7激活AKT信号传导的能力中起直接作用，而AKT信号传导又是MMP分泌最佳水平所需的。这些结果表明，E7 CKII磷酸受体位点因此继续对E7在源自宫颈癌的细胞中的活性起重要作用，并表明阻断E7的这种活性可预期具有治疗潜力。
The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.