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 Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.     

PATJ, a tight junction-associated PDZ protein, is a novel degradation target of high-risk human papillomavirus E6 and the alternatively spliced isoform 18 E6

The E6 protein from high-risk human papillomavirus types interacts with and degrades several PDZ domain-containing proteins that localize to adherens junctions or tight junctions in polarized epithelial cells. We have identified the tight junction-associated multi-PDZ protein PATJ (PALS1-associated TJ protein) as a novel binding partner and degradation target of high-risk types 16 and 18 E6. PATJ functions in the assembly of the evolutionarily conserved CRB-PALS1-PATJ and Par6-aPKC-Par3 complexes and is critical for the formation of tight junctions in polarized cells. The ability of type 18 E6 full-length to bind to, and the subsequent degradation of, PATJ is dependent on its C-terminal PDZ binding motif. We demonstrate that the spliced 18 E6* protein, which lacks a C-terminal PDZ binding motif, associates with and degrades PATJ independently of full-length 18 E6. Thus, PATJ is the first binding partner that is degraded in response to both isoforms of 18 E6. The ability of E6 to utilize a non-E6AP ubiquitin ligase for the degradation of several PDZ binding partners has been suggested. We also demonstrate that 18 E6-mediated degradation of PATJ is not inhibited in cells where E6AP is silenced by shRNA. This suggests that the E6-E6AP complex is not required for the degradation of this protein target.     

Regulation of nuclear receptor activities by two human papillomavirus type 18 oncoproteins,E6 and E7

The human papillomavirus (HPV) E6 and E7 oncoproteins are two major proteins that remain expressing in HPV-associated human cancers. The high-risk HPVs synthesize E6 and E7 oncoproteins to alter the function of cellular regulatory proteins, such as p53 and retinoblastoma gene product, respectively. In this study, we demonstrated that HPV-18 E6 and E7 proteins were able to directly interact with some nuclear receptors (NRs), such as thyroid receptor, androgen receptor, and estrogen receptor (ER), whether or not appropriate hormones were present. The functional roles of these two oncoproteins in NRs depended on the cell type (including ligand), promoter context, and NR type. These two oncoproteins regulated ER functions through ER's AF-1, AF-2, or both. Hence, our results provide new insights into the mechanisms controlling the proliferation and immortalization of HPV infected cells by these two oncoproteins mediating through their regulatory functions in NR systems.     

Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule.

Claudins, most of which end in valine at their COOH termini, constitute tight junction (TJ) strands, suggesting that TJ strands strongly attract PDZ-containing proteins. Indeed, ZO-1, -2, and -3, each of which contains three PDZ domains, were shown to directly bind to claudins. Using the yeast two-hybrid system, we identified ZO-1 and MUPP1 (multi-PDZ domain protein 1) as binding partners for the COOH terminus of claudin-1. MUPP1 has been identified as a protein that contains 13 PDZ domains, but it has not been well characterized. In vitro binding assays with recombinant MUPP1 confirmed the interaction between MUPP1 and claudin-1 and identified PDZ10 as the responsible domain for this interaction. A polyclonal antibody specific for MUPP1 was then generated. Immunofluorescence confocal microscopy as well as immunoelectron microscopy with this antibody revealed that in polarized epithelial cells MUPP1 was exclusively concentrated at TJs. Furthermore, in vitro binding and transfection experiments showed that junctional adhesion molecule, another TJ adhesion molecule, also bound to the PDZ9 domain of MUPP1. These findings suggested that MUPP1 is concentrated at TJs in epithelial cells through its binding to claudin and junctional adhesion molecule and that it may function as a multivalent scaffold protein that recruits various proteins to TJs.     

Human papillomavirus type 18 E1 protein is translated from polycistronic mRNA by a discontinuous scanning mechanism

Papillomaviruses are small double-stranded DNA viruses that replicate episomally in the nuclei of infected cells. The full-length E1 protein of papillomaviruses is required for the replication of viral DNA. The viral mRNA from which the human papillomavirus type 18 E1 protein is expressed is not known. We demonstrate that in eukaryotic cells, the E1 protein is expressed from polycistronic mRNA containing E6, E7, and E1 open reading frames (ORFs). The translation of adjacent E7 and E1 ORFs is not associated; it is performed by separate populations of ribosomes. The translation of the downstream E1 gene is preceded by ribosome scanning. Scanning happens at least at the 5' end of the polycistronic mRNA and also approximately 100 bp in front of the E1 gene. Long areas in middle of the mRNA are bypassed by ribosomes, possibly by ribosomal "shunting." Inactivation of short minicistrons in the upstream area of the E1 gene did not change the expression level of the E1 gene.     

The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.     

Targeting the human papillomavirus E6 and E7 oncogenes through expression of the bovine papillomavirus type 1 E2 protein stimulates cellular motility

Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.     

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.     

Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or lowrisk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA. Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.     

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.     

Mechanisms of tumor suppressor protein inactivation by the human papillomavirus E6 and E7 oncoproteins

There are now several examples where experimental and epidemiologic data have implied a causative role for viruses in human cancer. Human papillomavirus (HPV) DNA is found in approximately 90% of cervical cancers. Only a subset of the HPV types that infect the anogential tissues, however, are associated with cancer. Interestingly, only the cloned DNA of this subset is capable of immortalizing human primary genital keratinocytes in culture. The oncoproteins of the HPVs are encoded by the E6 and E7 genes. Analogous to the oncoproteins of certain other DNA tumor viruses, the E6 and E7 proteins have been shown to functionally inactivate the tumor suppressor proteins p53 and pRB, respectively. We will review what is known of the mechanisms by which the E6 and E7 proteins inactivate these tumor suppressors and the evidence that these activities are related to the transforming capabilities of the HPVs associated with cancer.     

The PDZ ligand domain of the human papillomavirus type 16 E6 protein is required for E6's induction of epithelial hyperplasia in vivo

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6(WT)) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6(Delta 146-151), from the human keratin 14 (K14) promoter. The K14E6(Delta 146-151) mice exhibit a radiation response similar to that of the K14E6(WT) mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6(Delta 146-151) mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia.     

A new crucial protein interaction element that targets the adenovirus E4-ORF1 oncoprotein to membrane vesicles

Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, including MUPP1, PATJ, MAGI-1, ZO-2, and Dlg1. Nevertheless, because certain E4-ORF1 mutations that alter neither the sequence nor the function of the PBM abolish E4-ORF1-induced PI3K activation and cellular transformation, we reasoned that E4-ORF1 must possess an additional crucial protein element. In the present study, we identified seven E4-ORF1 amino acid residues that define this new element, designated domain 2, and showed that it mediates binding to a 70-kDa cellular phosphoprotein. We also discovered that domain 2 or the PBM independently promotes E4-ORF1 localization to cytoplasmic membrane vesicles and that this activity of domain 2 depends on E4-ORF1 trimerization. Consistent with the latter observation, molecular-modeling analyses predicted that E4-ORF1 trimerization brings together six out of seven domain 2 residues at each of the three subunit interfaces. These findings importantly demonstrate that PI3K activation and cellular transformation induced by E4-ORF1 require two separate protein interaction elements, domain 2 and the PBM, each of which targets E4-ORF1 to vesicle membranes in cells.     

Identification of a G(2) arrest domain in the E1 wedge E4 protein of human papillomavirus type 16

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1 wedge E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1 wedge E4 arrests HeLa cervical epithelial cells in G(2). 16E1 wedge E4 also caused a G(2) arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1 wedge E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1 wedge E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 wedge E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 wedge E4-mediated G(2) arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G(2) arrest was also mediated by the E1 wedge E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1 wedge E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G(2) arrest. We conclude that, like other papillomavirus proteins, 16E1 wedge E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.     

The interaction between human papillomavirus type 16 and FADD is mediated by a novel E6 binding domain

High-risk strains of human papillomavirus, such as types 16 and 18, have been etiologically linked to cervical cancer. Most cervical cancer tissues are positive for both the E6 and E7 oncoproteins, since it is their cooperation that results in successful transformation and immortalization of infected cells. We have reported that E6 binds to tumor necrosis factor receptor 1 and to Fas-associated death domain (FADD) and, in doing so, prevents E6-expressing cells from responding to apoptotic stimuli. The binding site of E6 to FADD localizes to the first 23 amino acids of FADD and has now been further characterized by the use of deletion and site-directed mutants of FADD in pull-down and functional assays. The results from these experiments revealed that mutations of serine 16, serine 18, and leucine 20 obstruct FADD binding to E6, suggesting that these residues are part of the E6 binding domain on FADD. Because FADD does not contain the two previously identified E6 binding motifs, the LxxLsh motif, and the PDZ motif, a novel binding domain for E6 has been identified on FADD. Furthermore, peptides that correspond to this region can block E6/FADD binding in vitro and can resensitize E6-expressing cells to apoptotic stimuli in vivo. These results demonstrate the existence of a novel E6 binding domain.     

Link of the unique oncogenic properties of adenovirus type 9 E4-ORF1 to a select interaction with the candidate tumor suppressor protein ZO-2

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region-encoded ORF1 (E4-ORF1) protein. We report here that the PDZ domain-containing protein ZO-2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4-ORF1 but not for non-tumorigenic wild-type E4-ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C-terminal PDZ domain-binding motif of Ad9 E4- ORF1 and the first PDZ domain of ZO-2, and in cells this interaction resulted in aberrant sequestration of ZO-2 within the cytoplasm. Furthermore, transformation-defective Ad9 E4-ORF1 mutants exhibited impaired binding to and sequestration of ZO-2 in cells, and overexpression of wild-type ZO-2, but not mutant ZO-2 lacking the second and third PDZ domains, interfered with Ad9 E4-ORF1-induced focus formation. Our results suggest that the select capacity to complex with the candidate tumor suppressor protein ZO-2 is key to defining the unique transforming and tumorigenic properties of the Ad9 E4-ORF1 oncoprotein.