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.
The high-risk Human Papillomavirus (HPV) E6 oncoproteins are characterised by the presence of a class I PDZ-binding motif (PBM) on their extreme carboxy termini. The PBM is present on the E6 proteins ...derived from all cancer-causing HPV types, but can also be found on some related non-cancer-causing E6 proteins. We have therefore been interested in investigating the potential functional differences between these different E6 PBMs. Using an unbiased proteomic approach in keratinocytes, we have directly compared the interaction profiles of these different PBMs. This has allowed us to identify the potential PDZ target fingerprints of the E6 PBMs from 7 different cancer-causing HPV types, from 3 HPV types with weak cancer association, and from one benign HPV type that possesses an ancestral PBM. We demonstrate a striking increase in the number of potential PDZ targets bound by each E6 PBM as cancer-causing potential increases, and show that the HPV-16 and HPV-18 PBMs have the most flexibility in their PDZ target selection. Furthermore, the specific interaction with hScrib correlates directly with increased oncogenic potential. In contrast, hDlg is bound equally well by all the HPV E6 PBMs analysed, indicating that this is an evolutionarily conserved interaction, and was most likely one of the original E6 PBM target proteins that was important for the occupation of a potential new niche. Finally, we present evidence that the cell junction components ZO-2 and β-2 syntrophin are novel PDZ domain-containing targets of a subset of high-risk HPV types.
Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators ...of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.
This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.
The cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins target a number of cellular proteins that contain PDZ domains. However, the role of many of these interactions in either the ...HPV life cycle or in HPV-induced malignancy remains to be defined. Previous studies had shown that MAGI-1 was one of the most strongly bound PDZ domain-containing substrates of E6, and one consequence of this interaction appeared to facilitate the perturbation of tight junctions (TJs) by E6. In this study, we describe the generation of a mutation, K499E, within the MAGI-1 PDZ1 domain, which is resistant to E6 targeting. This mutant allows restoration of MAGI-1 expression in HPV-positive cells and defines additional activities of MAGI-1 that are overcome as a consequence of the association with E6. The reexpression of MAGI-1 in HPV-positive cells results in an increased recruitment of ZO-1 and PAR3 to sites of cell-cell contact, repression of cell proliferation, and induction of apoptosis. While the K499E mutation does not significantly affect these intrinsic activities of MAGI-1 in HPV-negative cells, its resistance to E6 targeting in an HPV-positive setting results in more cells expressing the mutant MAGI-1 than the wild-type MAGI-1, with a corresponding increase in TJ assembly, induction of apoptosis, and reduction in cell proliferation. These studies provide compelling evidence of a direct role for the perturbation of MAGI-1 function by E6 in the HPV life cycle and in HPV-induced malignancy.
It is clear that the targeting of PDZ-containing substrates by E6 is important for the normal viral life cycle and for the progression to malignancy. Nevertheless, which of these PDZ domain-containing proteins is relevant for HPV pathology is still elusive. In a previous study, we provided evidence that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins; however, the biological consequences associated with loss of MAGI-1 expression in HPV-positive cervical cancer cells are still poorly understood. Using a mutant MAGI-1, resistant to E6-mediated degradation, we show that its expression in cervical cancer cells promotes membrane recruitment of the tight junction-associated proteins ZO-1 and PAR3, represses cell proliferation, and promotes apoptosis. These findings suggest that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis.
Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to ...confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.
A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute ...directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.
Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to ...be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.
Protein kinase B (PKB) or Akt is one of several second messenger kinases that are activated by cell attachment and growth factor signaling, and that transmit signals to the cell nucleus to inhibit ...apoptosis and thereby increase cell survival during proliferation. Other viral proteins target this pathway by increasing PKB/Akt phosphorylation, and this pathway has been implicated in the transformation of human keratinocytes by HPV E6 and E7, together with activated notch 1. Here, we examine how HPV E7 expression affects the phosphorylation of PKB. We show that HPV-16 E7 increases the level of phosphorylation of PKB in response to serum stimulation, by a mechanism independent of downregulation of PTEN phosphatase, a known inhibitor of the PI3K (PI3 kinase) pathway. The use of specific antibodies shows that some proportion of PKB/Akt that is phosphorylated both on threonine 308 and serine 473 is maintained in the presence of E7 in a PI3 kinase-independent manner, and is activated for phosphorylation of BAD, a known downstream target of PKB/Akt. Use of E7 mutants has ruled out both an inhibition of IGFBP-3, a known E7 target and PKB/Akt modulator, and the interaction of E7 with cellular pocket proteins, as being the mechanism for the PKB/Akt stimulation. PKB binds PP2A and is a known substrate of PP2A. Here, we show that HPV E7 also binds to both the 35 kDa catalytic and 65 kDa structural subunits of PP2A, an interaction that sequesters these subunits and inhibits their interaction with PKB, thereby maintaining PKB/Akt signaling by inhibiting its dephosphorylation.
Human papillomavirus (HPV) E7 plays a major role in HPV-induced malignancy, perturbing cell cycle regulation, and driving cell proliferation. Major targets of cancer-causing HPV E7 proteins are the ...pRB family of tumor suppressors, which E7 targets for proteasome-mediated degradation and whose interaction is promoted through an acidic patch, downstream of the LXCXE motif in E7, that is subject to phosphorylation by casein kinase II (CKII). In this study we show that HPV-16 E7 targets the AP2-complex, which plays a critical role in cargo recognition in clathrin-mediated endocytosis. Intriguingly, HPV-16 E7 contains a specific amino acid sequence for AP2 recognition, and this overlaps the pRb LXCXE recognition sequence but involves completely different amino acid residues. HPV-16 E7 does this by binding to the AP2-μ2 adaptor protein subunit via residues 25-YEQL-28 within the LXCXE motif. Point mutations at Y25 within 22-LYCYE-26 suggest that the interaction of E7 with AP2-μ2 is independent from pRB binding. In cells, this interaction is modulated by acidic residues downstream of LXCXE, with the binding being facilitated by CKII-phosphorylation of the serines at positions 31 and 32. Finally, we also show that association of HPV-16 E7 with the AP2 adaptor complex can contribute to cellular transformation under low-nutrient conditions, which appears to be mediated, in part, through inhibition of AP2-mediated internalization of epidermal growth factor receptor (EGFR). This indicates that E7 can modulate endocytic transport pathways, with one such component, EGFR, most likely contributing toward the ability of E7 to induce cell transformation and malignancy. These studies define a new and unexpected role for HPV-16 E7 in targeting clathrin-mediated endocytosis.
Despite being a very small protein, HPV-E7 has a wide range of functions within the infected cell, many of which can lead to cell transformation. High-risk HPV-E7 deregulates the function of many cellular proteins, perturbing cellular homeostasis. We show that a novel target of HPV-E7 is the clathrin-adaptor protein 2 complex (AP2) μ2 subunit, interacting via residues within E7's pRB-binding region. Mutational studies show that an AP2 recognition motif is present in the CR2 region and is conserved in >50 HPV types, suggesting a common function for this motif in HPV biology. Mutational analysis suggests that this motif is important for cellular transformation, potentially modulating endocytosis of growth factor receptors such as EGFR, and thus being a novel activity of E7 in modulating clathrin-mediated endocytosis and cargo selection. This study has important implications for the molecular basis of E7 function in modulating protein trafficking at the cell surface.
The components of the Scrib/Dlg tumour suppressor complex have complementary roles in Drosophila and loss of both proteins is a common event in many different human tumours. However no studies have ...directly addressed the respective contributions of loss of hScrib and hDlg in the same human cell background to cellular phenotypes associated with cell transformation. In human HaCaT keratinocytes we show that removal of hScrib greatly reduces cell-cell contact and cell-matrix interactions, and promotes an invasive phenotype. Conversely, in cells lacking hDlg1 cell-cell contacts are maintained and there are decreases in both cell growth and invasion. However, hDlg-depleted cells show increased resistance to a specialized form of apoptosis known as anoikis, to which cells lacking hScrib are highly susceptible. Thus whilst it has been widely assumed that hScrib and hDlg have complementary roles, these studies in fact demonstrate that hScrib and hDlg1 have distinct and opposing functions in human keratinocytes.
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