E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling

Diacylglycerol kinase zeta is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase zeta has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase zeta interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase zeta-interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase zeta. Because diacylglycerol kinase zeta is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase zeta regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase zeta showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase zeta-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase zeta in the modulation of membrane trafficking, which may also help to define SNX27 function.     

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

Phosphorylation of SNX27 by MAPK11/14 links cellular stress–signaling pathways with endocytic recycling

Endocytosed proteins can be delivered to lysosomes for degradation or recycled to either the trans-Golgi network or the plasma membrane. It remains poorly understood how the recycling versus degradation of cargoes is determined. Here, we show that multiple extracellular stimuli, including starvation, LPS, IL-6, and EGF treatment, can strongly inhibit endocytic recycling of multiple cargoes through the activation of MAPK11/14. The stress-induced kinases in turn directly phosphorylate SNX27, a key regulator of endocytic recycling, at serine 51 (Ser51). Phosphorylation of SNX27 at Ser51 alters the conformation of its cargo-binding pocket and decreases the interaction between SNX27 and cargo proteins, thereby inhibiting endocytic recycling. Our study indicates that endocytic recycling is highly dynamic and can crosstalk with cellular stress–signaling pathways. Suppression of endocytic recycling and enhancement of receptor lysosomal degradation serve as new mechanisms for cells to cope with stress and save energy.     

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.     

A systematic analysis of human papillomavirus (HPV) E6 PDZ substrates identifies MAGI-1 as a major target of HPV type 16 (HPV-16) and HPV-18 whose loss accompanies disruption of tight junctions

The E6 proteins from high-risk, cancer-causing types of human papillomavirus (HPV) are characterized by the presence of a PDZ (PSD95/Dlg/ZO-1) binding motif in their extreme carboxy termini, through which they interact with a number of cellular PDZ domain-containing substrates. In order to ascertain how many of these are degraded by E6 in vivo, we performed an extensive analysis of the effects of E6 ablation on the expression levels of a number of previously reported E6 PDZ substrates. Using HPV type 16 (HPV-16)-positive CaSKi cells and HPV-18-positive HeLa cells, we have found that MAGI-1 is a major degradation target of both HPV-16 and HPV-18 E6. In contrast, hDlg, hScrib, PTPN3, TIP2, FAP1, and PSD95 all exhibit various degrees of susceptibility to E6-induced degradation, and a high degree of HPV type specificity is observed for certain substrates. We also show that E6 preferentially targets MAGI-1 within the nucleus and at membrane sites. One of the direct consequences of MAGI-1 degradation is a loss of tight-junction integrity, as determined by mislocalization of the tight-junction protein ZO-1. Ablation of E6 expression restores tight junctions, and this restoration is dependent on the presence of MAGI-1. These results demonstrate that oncogenic HPV E6 proteins disrupt cellular tight junctions through the degradation of MAGI-1, and they provide further evidence of how the PDZ binding potential of E6 can contribute to HPV-induced malignancy.     

Structures of a human papillomavirus (HPV) E6 polypeptide bound to MAGUK proteins: mechanisms of targeting tumor suppressors by a high-risk HPV oncoprotein

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.     

Deficiency of sorting nexin 27 (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate receptor 2C (NR2C)

Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.     

Induction of apoptosis in cervical carcinoma cells by peptide aptamers that bind to the HPV-16 E7 oncoprotein.

Human papillomaviruses (HPV) of the high-risk type are causally involved in human tumors, in particular cervical carcinoma. Expression of the viral oncogenes E6 and E7 is maintained in HPV-positive tumors, and it was shown that E6 and E7 of HPV-16 can immortalize human keratinocytes, the natural host cells of the virus. Expression of the viral genes is also required for maintenance of the transformed phenotype. The oncogenic activity of the E6 and E7 oncoproteins is mediated by their physical and functional interaction with cellular regulatory proteins. To knock out the function of the E7 protein in living cells, we have developed peptide aptamers with high specific binding activity for the E7 protein of HPV-16. We show here that E7-binding peptide aptamers induce programmed cell death (apoptosis) in E7-expressing cells, whereas E7-negative cells are not affected. Furthermore, E7-binding peptide aptamers induce apoptosis in HPV-16-positive tumor cells derived from cervical carcinoma. The data suggest that E7-binding peptide aptamers may be useful tools to specifically eliminate HPV-positive tumors.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

Role of the PDZ domain-binding motif of the oncoprotein E6 in the pathogenesis of human papillomavirus type 31

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP1. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. The presence of this motif only in the high-risk HPVs suggests its possible role in HPV-induced oncogenesis. To investigate the role of the PDZ domain-binding motif of E6 in the HPV life cycle, two mutant HPV31 genomes were constructed: E6ValDelta, with a deletion of the last amino acid residue of E6 (valine), and E6ETQVDelta, with a deletion of the entire PDZ domain-binding motif of E6 (ETQV). Three human foreskin keratinocyte (HFK) cell lines were established which maintained transfected wild-type HPV31 or either of two mutant genomes. Cells containing either of two mutant genomes were significantly retarded in their growth rates and reduced in their viral copy numbers compared to those transfected with wild-type genomes. Western analysis did not reveal any significant changes in the levels of PDZ proteins following stable transfection of any HPV31 genomes into HFKs. Although the E6ETQVDelta-transfected HFKs exhibited a pattern of morphological differentiation that appeared different from the HPV31 wild-type-transfected HFKs in organotypic raft cultures, immunohistochemical analysis failed to identify substantial changes in the differentiation-dependent membrane localization of hDlg proteins. These results suggest that binding of E6 to PDZ proteins modulates the early viral functions such as proliferation and maintenance of the viral copy number in undifferentiated cells.     

PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

High-risk human papillomavirus (HPV) E6 proteins have a C-terminal PDZ binding motif through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recent studies have suggested that the RING-containing ubiquitin-protein ligase PDZRN3 might also be an HPV E6 target. In this analysis, we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ- and proteasome-dependent manner and provide a connection between the HPV life cycle and differentiation-related STAT signaling.     

E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.     

The human papillomavirus type 16 E7 oncoprotein activates the Fanconi anemia (FA) pathway and causes accelerated chromosomal instability in FA cells

Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.     

Human papillomaviruses and the specificity of PDZ domain targeting

The human papillomavirus (HPV) E6 oncoprotein is fundamental to the ability of these viruses to induce human malignancy. A defining characteristic of the HPV E6 oncoproteins found in cancer-causing HPV types is the presence of a PDZ binding motif at their extreme C-terminus. Through this motif, E6 is able to interact with a large number of cellular proteins that contain PDZ domains. Many of these cellular proteins are involved in regulation of processes associated with the control of cell attachment, cell proliferation, cell polarity and cell signaling. How E6 targets multiple proteins containing the same recognition domain is still an open question. In this review, we highlight aspects of E6 function and biology that help to answer this question, and thereby provide insight into the role of these substrates during development of HPV-induced malignancy.     

Papillomavirus E6 PDZ Interactions Can Be Replaced by Repression of p53 To Promote Episomal Human Papillomavirus Genome Maintenance

Cancer-associated human papillomaviruses (HPVs) express E6 oncoproteins that target the degradation of p53 and have a carboxy-terminal PDZ ligand that is required for stable episomal maintenance of the HPV genome. We find that the E6 PDZ ligand can be deleted and the HPV genome stably maintained if cellular p53 is inactivated. This indicates that the E6-PDZ interaction promotes HPV genome maintenance at least in part by neutralization of an activity that can arise from residual undegraded p53.