Human scribble, a novel tumor suppressor identified as a target of high-risk HPV E6 for ubiquitin-mediated degradation, interacts with adenomatous polyposis coli

Recently, we have identified human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, as a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing leucine-rich repeats and PDZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), which is a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. Here, we investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localized with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC caused disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC.     

The stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.     

Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for ∼ 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.     

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.     

HPV16 E6 augments Wnt signaling in an E6AP-dependent manner

In this study we investigated the effect of HPV16 E6 on the Wnt/β-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/β-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3β or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of β-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.     

Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes

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, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg.     

E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.     

hScrib, a human homologue of Drosophila neoplastic tumor suppressor, is a novel death substrate targeted by caspase during the process of apoptosis

hScrib, human homologue of Drosophila neoplastic tumor suppressor, was identified as a target of human papillomavirus E6 oncoprotein for the ubiquitin-mediated degradation. Here, we report that hScrib is a novel death substrate targeted by caspase. Full-length hScrib was cleaved by caspase during death ligands-induced apoptosis, which generates a p170 C-terminal fragments in Hela cells. In vitro cleavage assay using recombinant caspases showed that hScrib is cleaved by the executioner caspases. DNA damage-induced apoptosis caused loss of expression of full-length hScrib, which was recovered by addition of capase-3 inhibitor in HaCat cells. TUNEL positive apoptotic cells, which were identified 4 h after UV irradiation in HaCat cells, showed loss of hScrib expression at the adherens junction. Mutational analysis identified the caspase-dependent cleavage site of hScrib at the position of Asp-504. Although MDCK cells transfected with GFP-fused wild-type hScrib showed loss of E-cadherin expression and shrinkage of cytoplasm by UV irradiation, cells transfected with hScrib with Ala substitution of Asp-504 showed resistance to caspase-dependent cleavage of hScrib and intact expression of E-cadherin. These results indicate that caspase-dependent cleavage of hScrib is a critical step for detachment of cell contact during the process of apoptosis.     

E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6

We previously found that HPV16 E6 causes the degradation of the tumor suppressor protein TSC2, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. In the present study, we investigated the role of E6-associated protein (E6AP) in HPV16 E6-induced TSC2 degradation. Our results demonstrated that TSC2 was targeted for degradation in the presence or absence of HPV16 E6. Over-expression of E6AP enhanced the degradation of TSC2 by HPV16 E6, while expression of a dominant negative E6AP (C833A) inhibited the E6-induced degradation. Additionally, by using shRNAs to block E6AP expression in HPV16 positive and negative cells, we found a significantly prolonged TSC2 half-life. An in vivo ubiquitination assay was done to reveal that E6AP promoted the ubiquitination of TSC2 independent of HPV16 E6. We further found that TSC2 bound E6AP in the presence as well as in the absence of HPV16 E6. The binding regions on E6AP and TSC2 have been identified as amino acid (aa) 260–316, aa 428–500 and aa 1–175, aa 1251–1807, respectively. Taken together, degradation of TSC2 is mediated by E6AP ubiquitin ligase.     

Association of E6AP (UBE3A) with human papillomavirus type 11 E6 protein

The cellular E3 ubiquitin ligase E6AP (UBE3A) interacts with the cancer-associated HPV E6 oncoproteins, where together with the viral E6 oncoprotein it binds and targets the degradation of the p53 tumor suppressor. We find that the HPV-11E6 protein also associates with E6AP in vivo, and thereby can target the degradation of an E6-associated protein. Mutation of an E6-binding LXXLL peptide motif on E6AP eliminated the association, revealing a common mode of interaction between high- and low-risk E6 proteins and E6AP. E6AP was required for the in vivo degradation of DLG1 by both HVP-18 E6 and a chimeric HPV-11E6. The common functional interaction of both cancer-associated and non-cancer-associated E6 proteins with E6AP establishes a common mechanism for E6 proteins trophic to mucosal squamous epithelium.     

Human scribble accumulates in colorectal neoplasia in association with an altered distribution of beta-catenin

The loss of epithelial polarity and tissue architecture is a diagnostic feature of malignant tumors. In Drosophila, genetic studies identified 3 neoplastic tumor suppressor genes (nTSGs), and a loss of nTSGs has been shown to result in a disruption of apical-basal polarity and neoplastic growth in epithelial cells. Scribble is one type of the Drosophila nTSGs, which encodes a membrane-associated cytoplasmic protein containing the multi-PDZ domain. In contrast to Drosophila scribble, the oncogenic roles of its mammalian homologues have not yet been established. We herein immunohistochemically examined the distributions of hScrib protein in human colorectal neoplasia using affinity-purified antibody. In 50 cases of colorectal adenomas and adenocarcinomas, the accumulation of hScrib protein was commonly observed in comparison with the adjacent normal epithelia. Furthermore, the overexpression and distribution of hScrib was observed to extensively overlap with the cytoplasmic accumulation of beta-catenin. Like beta-catenin, the intense immunoreactivity of hScrib was often observed in small adenomas, thus, suggesting that hScrib could be involved in an early step of colon carcinogenesis. Five corresponding liver metastases showed a comparable immunoreactivity for anti-hScrib in comparison with their primary sites. In an immunofluorescence analysis on cultured cell lines, the loss of membranous staining of hScrib was observed according to the cytoplasmic translocation of beta-catenin. We herein demonstrate that the accumulation of hScrib protein might therefore be involved in colon carcinogenesis while also providing a possible link between hScrib and beta-catenin.     

The role of TP53 in Cervical carcinogenesis

Functional loss of the tumor suppressor p53 by alterations in its TP53 gene is a frequent event in cancers of different anatomical regions. Cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types. The viral oncoprotein E6 has the ability to associate with and neutralize the function of p53. E6 interacts with a 100-kDa cellular protein, termed E6 associated protein (E6AP; also called ubiquitin-protein ligase E3A or UBE3A), which functions as an ubiquitin protein ligase. The dimeric complex then binds p53 and E6AP catalyzes multi-ubiquitination and degradation of p53. The ability to promote p53 degradation is an exclusive property of E6 from the high-risk HPV types. Indeed, the low-risk E6 proteins lack this activity, although they can bind p53. Consistent with the E6 function of the high-risk HPV types, the majority of cervical cancer cells have a wild-type p53 gene, but the protein levels are strongly decreased. Several independent studies have shown that in a small percentage of cervical tumors the p53 gene is mutated. However, this event appears to be unrelated to the presence or absence of HPV infection and the nature of the tumor.     

非对称性分裂调节因子hScrib和hDlg与恶性肿瘤

肿瘤干细胞的鉴定成功对肿瘤发生的观点产生了新的挑战。干细胞最大的特点是非对称性分裂从而实现自我更新,如果这一过程被扰乱则会发生肿瘤。肿瘤抑制基因hscrib、hdlg被认为在这一分子机制中调节命运决定子在子代细胞中的分配。细胞极性是上皮组织的一个标志性特点,极性丢失与上皮的恶性转化密切相关。研究表明hScrib、hDlg是维持正常上皮极性的关键因子,在多种上皮性恶性肿瘤的发生中可能起一定作用。     

Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53

E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins. BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL. E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation. Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP. In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex. 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53. Such 16E6 mutations were typically within the amino-terminus of 16E6. Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6. However, the interaction of 16E6 with E6AP was complex. While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation. In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation. These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way. These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.     

人乳头瘤病毒16型修饰后E6E7基因免疫原性增强

利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7)，研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后，插人真核表达质粒获得pVRl012-fmE6E7，瞬时转染Cos-7细胞，免疫荧光法检测证实其表达后，在C57BL／6小鼠后腿肌肉进行裸DNA免疫，5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte，CTL)，间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应，与单独野生型E7基因免疫相比，E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。     

Cellular steady-state levels of "high risk" but not "low risk" human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53- and E6AP-binding capabilities

The group of mucosal epithelia-infecting human papillomaviruses (HPV) can be subdivided in "low" and "high risk" HPV types. Both types induce benign neoplasia (condyloma), but only the infection with a "high risk" HPV type is causally associated with an increased risk of developing anogenital tumors. The oncogenic potential of high risk HPVs resides at least partially in the viral E6 protein. The E6 protein targets the cellular p53 protein for proteasome-dependent degradation, which is associated with the immortalizing and transforming functions of these viruses. Recently the E6-dependent proteasome-mediated destabilization of additional cellular proteins (E6TP1, c-myc, Bak, hMCM7, human scribble, E6AP, MAGI-1) has been described, but the cellular mechanisms controlling the viral E6 protein stability itself have been so far not analyzed. In this study, we transiently expressed the E6 genes of the high risk HPV type 16, the low risk HPV types 6a and 11, and the cutaneous epithelia-infecting HPV types 5 and 8 from a eucaryotic expression vector and compared the cellular steady-state levels of the expressed E6 proteins. We demonstrated that the high risk HPV 16 E6 protein possesses the lowest steady-state level in comparison to the low risk HPV type E6 proteins and the cutaneous epithelia-infecting HPV type E6 proteins. Inhibition of cellular proteasome-dependent protein degradation led to an increase in steady-state levels of high risk but not of low risk E6 proteins. Analysis of functionally deficient HPV 16 E6 proteins in p53 null- and p53 wild-type-expressing cell lines revealed that the cellular steady-state level of this protein is influenced neither by its p53- nor its E6AP-binding abilities.     

Microarray analysis identifies differentiation-associated genes regulated by human papillomavirus type 16 E6

In this study, we used oligonucleotide microarray analysis to determine which cellular genes are regulated by the human papillomavirus type 16 (HPV-16) E6 oncoprotein. We found that E6 causes the downregulation of a large number of cellular genes involved in keratinocyte differentiation, including genes such as small proline-rich proteins, transglutaminase, involucrin, elafin, and cytokeratins, which are normally involved in the production of the cornified cell envelope. In contrast, E6 upregulates several genes, such as vimentin, that are usually expressed in mesenchymal lineages. E6 also modulates levels of genes involved in inflammation, including Cox-1 and Nag-1. By using E6 mutants that differentially target p53 for degradation, we determined that E6 regulates cellular genes by both p53-dependent and independent mechanisms. The microarray data also indicate that HPV-16 E6 modulates certain effects of HPV-16 E7 on cellular gene expression. The identification of E6-regulated genes in this analysis provides a basis for further studies on their role in HPV infection and cellular transformation.     

Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle

The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another alpha-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span.     

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.     

Stabilization of HPV16 E6 protein by PDZ proteins, and potential implications for genome maintenance

The E6 protein from high-risk human papillomaviruses appears necessary for persistence of viral episomes in cells but the underlying mechanism is unclear. E6 has many activities, including its ability to bind and degrade PDZ domain-containing proteins, such as hScrib. However little is known about the role of these interactions for E6 function and the viral life cycle. We now show that the levels of expression of wild-type E6 are increased in the presence of hScrib whilst a mutant E6 protein lacking the PDZ-binding motif is found at lower levels as it is turned over more rapidly by the proteasome. This correlates with an inability of genomes containing this mutation to be maintained as episomes. These results show that E6 association with certain PDZ domain-containing proteins can stabilize the levels of E6 expression and provides one explanation as to how the PDZ-binding capacity of E6 might contribute to genome episomal maintenance.