Research Article: The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells

Date Published: May 22, 2019

Publisher: Public Library of Science

Author(s): Om Basukala, Suruchi Mittal, Paola Massimi, Marco Bestagno, Lawrence Banks, Robert F. Kalejta.


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.

Partial Text

Human papillomaviruses (HPVs) are major causes of human cancer, with cervical cancer being the most important. Whilst there are over 200 different HPV types, only a small subset are responsible for the development of human cancers and, of these, HPV-16 and HPV-18 are the most common [1]. HPVs replicate in differentiating epithelia, in cells that would normally have exited the cell cycle. Since HPVs do not encode any proteins that can be used to replicate DNA, they need to drive these non-dividing cells back into cell cycle, so that the viral DNA can be amplified. This is brought about by the action of the two viral oncoproteins, E6 and E7, which together create an environment favourable for viral DNA replication [2]. This is achieved primarily through interfering with cellular growth control pathways, with E7 targeting many elements involved in the control of cell cycle, whilst E6 inhibits the pro-apoptotic response of the cell to this unscheduled DNA replication [3, 4]. In rare instances, the viral life cycle is perturbed and the events that, ultimately, give rise to malignancy are initiated. In these tumour-derived cell lines, E6 and E7 continue to be expressed, and loss of expression of either brings about cessation of cell growth and the induction of apoptosis [5–7]. Therefore, both proteins are excellent targets for therapeutic intervention in HPV-induced malignancy.

Previous studies have shown that HPV-16 E7 is phosphorylated at residues S31 and S32 by CKII, and protein alignments would also suggest that the equivalent residues S32 and S34 in HPV-18 E7 would likewise be similarly phosphorylated by CKII [14]. In order to confirm this, we first performed a series of in vitro phosphorylation assays on purified GST-18 E7, using commercially available purified CKII and 32P-γATP. To verify the identity of the phospho-acceptor site, we also generated single and double amino acid substitutions within the putative CKII phospho-acceptor site of HPV-18 E7. The results obtained are shown in Fig 1 and demonstrate that both S32 and S34 are phosphorylated by CKII. Interestingly, the single S32A mutation appears to increase the overall level of E7 phosphorylation, although the reasons for this are unclear. Mutation of both of these residues completely abolishes CKII phosphorylation of E7, confirming that these are the only CKII phospho-acceptor sites in HPV-18 E7. Taken together, these results demonstrate that both S32 and S34 are phosphorylated by CKII.

Despite extensive characterization of the different biochemical functions of the HPV E6 and E7 oncoproteins, and their respective contributions towards the development of cell transformation and malignancy, there is still very little information on the continued requirement for specific activities of E6 and E7 in cells derived from a cervical cancer. This is important, since we know there is an absolute requirement for both oncoproteins for maintaining the transformed phenotype [5–7], but which specific biochemical functions of E6 and E7 contribute towards this remains obscure. In this study we have begun to address this question by performing genome editing of the HPV-18 E7 CKII phospho-acceptor site in cervical cancer-derived C4-1 cells. We find that an intact CKII phospho-acceptor site in E7 remains important, both for optimal levels of cell proliferation and for maintaining high levels of invasive potential.




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