Targeting E3 Research Articles

P53-Independent Abrogation of a Postmitotic Checkpoint Contributes to Human Papillomavirus E6-Induced Polyploidy

Polyploidy is often an early event during cervical carcinogenesis, and it predisposes cells to aneuploidy, which is thought to play a causal role in tumorigenesis. Cervical and anogenital cancers are induced by the high-risk types of human papillomavirus (HPV). The HPV E6 oncoprotein induces polyploidy in human keratinocytes, yet the mechanism is not known. It was believed that E6 induces polyploidy by abrogating the spindle checkpoint after mitotic stress. We have tested this hypothesis using human keratinocytes in which E6 expression induces a significant amount of polyploidy. We found that E6 expression does not affect the spindle checkpoint. Instead, we provide direct evidence that E6 is capable of abrogating the subsequent G(1) arrest after adaptation of the mitotic stress. E6 targets p53 for degradation, and previous studies have shown an important role for p53 in modulation of the G(1) arrest after mitotic stress. Importantly, we have discovered that E6 mutants defective in p53 degradation also induce polyploidy, although with lower efficiency. These results suggest that E6 is able to induce polyploidy via both p53-dependent and p53-independent mechanisms. Therefore, our studies highlight a novel function of HPV E6 that may contribute to HPV-induced carcinogenesis and improve our understanding of the onset of the disease.

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.

High-Risk HPV E6 Oncoproteins Assemble into Large Oligomers that Allow Localization of Endogenous Species in Prototypic HPV-Transformed Cell Lines

The E6 oncoproteins of high-risk HPV types 16 and 18 are involved in the development of cervical cancer. Besides its determinant role in carcinogenic progression, HPV E6 oncoprotein has also been instrumental in elucidating fundamental aspects of p53 function and its ubiquitin-proteasome degradation, with counterpart activities in various DNA tumor viruses. Establishing the conformational state and cellular distribution unequivocally for the endogenous protein in HPV-transformed cell lines derived from carcinomas is essential for understanding the underlying mechanism. Recombinant E6 from high-risk strains 16 and 18 folds into soluble oligomers of approximately 1.2 MDa, which are thermostable and display cooperative loss of tertiary and secondary structure upon chemical denaturation. Antibodies raised against these assemblies locate E6 evenly distributed in the cells. By depleting the polyclonal serum by immunoblocking with monomeric E6, the nuclei of Hela and CaSki cells become completely devoid of label, indicating that monomeric species are mainly localized in the nucleus and that both monomers and oligomers share epitopes. The monomeric species promote degradation of p53 by the proteasome, which correlates with the nuclear localization we describe. In contrast, the oligomeric E6 does not promote p53 degradation, in agreement with its cytoplasmic localization inferred from the immunoneutralization experiments. Our results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.

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.

Critical Roles for Non-pRb Targets of Human Papillomavirus Type 16 E7 in Cervical Carcinogenesis

High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage-induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis.

Human papillomavirus type 16 E6 protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes E6-associated protein-mediated ubiquitination and proteasomal degradation

The PDZ proteins such as hDLG, hScrib and MAGIs function as the membrane-associated protein scaffolds and have been shown to interact with the high-risk human papillomavirus (HPV) E6s. In this report, we identify a Golgi-associated PDZ protein, cystic fibrosis transmembrane regulator-associated ligand (CAL) as a cellular target of HPV16 E6 by the proteomic approach. The carboxy-terminal PDZ-binding motif of HPV16 E6 specifically interacts with the PDZ domain of CAL, and the interaction enhances proteasome-mediated degradation of CAL. HPV16 E6 interacts with CAL more strongly and degrades it better than HPV18 E6 owing to the more compatible PDZ-binding motif. CAL is ubiquitinated by the E6/E6-associated protein (E6AP) complex or by E6AP alone, albeit less efficiently, which indicates that it could be a normal target of E6AP. Although it downregulates CAL at the transcript level, small interfering RNA-induced depletion of HPV16 E6 in Caski cells stabilizes CAL at the protein level, suggesting that HPV16 E6 mediates the proteasomal degradation of CAL in HPV-positive cervical cancer cells. HPV16 E6 may tightly regulate the vesicular trafficking processes by interacting with CAL, and such a modification can contribute to the development of cervical cancer.

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Peptide‐Specific Antibody as a Tool to Evaluate TRPM3 Ion Channel Function

Calcium channel blockers can be used to selectively inhibit calcium influx across a membrane, making them potentially useful as therapeutic tools. Existing transient receptor potential (TRP) channel blockers are relatively non-specific, but have previously been used to characterise individual members of the TRP family. The activation of TRP channels can be more specifically inhibited through the use of anti-peptide antibodies targeted to particular regions of the channel structure (Xu & Beech, 2001. Circ. Res. 88:84–87). The polyclonal antibody TM3E3 was designed to specifically bind to a peptide corresponding to the 3rd extracellular loop of TRPM3, a member of the melastatin-like TRP family. The location and sequence of the peptide were determined using hydrophilicity analysis of the channel sequence (Xu et al., 2005. Nat. Biotechnol. 23:1289–1293). Here we use Ca2+-imaging to study the specificity of TM3E3 for human TRPM3 expressed in HEK-293 cells. Extracellular application of the antibody has a functional effect, inhibiting the sphingosine-induced activation of TRPM3. It does not inhibit activation of the closely related channel TRPM2 or the canonical family member TRPC5. TM3E3 will serve as a useful tool for further characterisation of endogenous TRPM3 in a variety of cells and tissues, and more importantly the results strengthen the idea that E3 targeting may be used to generate specific ion channel blockers for a variety of channels, with therapeutic potential. This work is supported by the BBSRC, the Welcome Trust and the BHF. Many thanks to C. Harteneck for providing the TRPM3 clone.

How Dihydrolipoamide Dehydrogenase-binding Protein Binds Dihydrolipoamide Dehydrogenase in the Human Pyruvate Dehydrogenase Complex

The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-angstroms resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.

Proteasome inhibitor MG132 sensitizes HPV‐positive human cervical cancer cells to rhTRAIL‐induced apoptosis

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.

The Human DEK Proto-Oncogene Is a Senescence Inhibitor and an Upregulated Target of High-Risk Human Papillomavirus E7

The human DEK proto-oncogene is a nucleic acid binding protein with suspected roles in human carcinogenesis, autoimmune disease, and viral infection. Intracellular DEK functions, however, are poorly understood. In papillomavirus-positive cervical cancer cells, downregulation of viral E6/E7 oncogene expression results in cellular senescence. We report here the specific repression of DEK message and protein levels in senescing human papillomavirus type 16- (HPV16-) and HPV18-positive cancer cell lines as well as in primary cells undergoing replicative senescence. Cervical cancer cell senescence was partially overcome by DEK overexpression, and DEK overexpression was sufficient for extending the life span of primary keratinocytes, supporting critical roles for this molecule as a senescence regulator. In order to determine whether DEK is a bona fide HPV oncogene target in primary cells, DEK expression was monitored in human keratinocytes transduced with HPV E6 and/or E7. The results identify high-risk HPV E7 as a positive DEK regulator, an activity that is not shared by low-risk HPV E7 protein. Experiments in mouse embryo fibroblasts recapitulated the observed E7-mediated DEK induction and demonstrated that both basal and E7-induced regulation of DEK expression are controlled by the retinoblastoma protein family. Taken together, our results suggest that DEK upregulation may be a common event in human carcinogenesis and may reflect its senescence inhibitory function.

Generation of functional ion-channel tools by E3 targeting

Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of Na(V)1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.

Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600

The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.

Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A

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.

Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

Erythroid Inhibition by AML1-ETO: Repression of GATA-1 Function by a Novel Non-Transcriptional Mechanism Involving Targeted Protein Degradation.

AML1-ETO (A/E) is the fusion product of a chromosomal translocation, t(8;21) frequently associated with FAB M2 acute myeloid leukemia (AML). The fusion combines the runt domain of the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. Clinical cases of AML with t(8;21) are distinguished by blockade in erythroid differentiation. In addition, enforced expression of A/E in primary human erythroid progenitors impairs differentiation. Existing paradigms postulate that A/E exerts its leukemogenic effects through recruitment to RUNX binding sites of cofactors such as corepressors, histone deacetylases (HDACs), and DNA methyltransferases (DNMTs), causing repression of RUNX target genes. However, this paradigm fails to explain effects of A/E on erythropoiesis as erythroid genes generally lack functional RUNX sites. We have published a physical and functional interplay between RUNX1 and the erythroid master regulator GATA-1 (Blood 101:4333). Furthermore, A/E physically interacted and functionally interfered with GATA-1. In the current studies we have examined domain and cofactor requirements for A/E inhibition both of GATA-1 function and of erythroid differentiation. Deletional mutagenesis of A/E demonstrated that the zinc finger (NH4) and runt domains were absolutely required for GATA-1 inhibition. Treatment with HDAC and DNMT inhibitors failed to affect A/E repression of GATA-1. RNAi knockdown of all known NH4 interactors, HDACs 1-3, N-CoR, SMRT-A, and SMRT-B also failed to affect A/E inhibition of GATA-1. Inducible expression of A/E in MEL cells caused downregulation of endogenous GATA-1 protein and mRNA, an effect dependent on induction of erythroid differentiation. A coexpressed GATA-1-GFP fusion showed downregulation with identical kinetics to endogenous GATA-1. Interestingly, proteasome-specific inhibitors effectively prevented the downregulation of endogenous GATA-1 and GATA-1-GFP caused by induction of A/E coupled with erythroid differentiation. Fluorescence microscopy showed a striking relocation of GATA-1-GFP from the nucleus to discrete, paranuclear bodies upon joint induction of A/E expression and erythroid differentiation. Our findings indicate that A/E inhibition of GATA-1 occurs through a previously undescribed mechanism that involves GATA-1 redistribution to novel cellular structures followed by proteasome-mediated degradation. These findings expand the paradigm of A/E leukemogenicity to include a non-transcriptional mechanism in which a growth inhibitor/tumor suppressor, GATA-1, is targeted by A/E for proteolytic degradation in a manner reminiscent of human papilloma virus E6 targeting of p53 for degradation in cervical carcinogenesis.

Degradation of hDlg and MAGIs by human papillomavirus E6 is E6-AP-independent

An important characteristic of the E6 proteins derived from cancer-associated human papillomaviruses (HPVs) is their ability to target cellular proteins for ubiquitin-mediated degradation. Degradation of the p53 tumour suppressor protein by E6 is known to involve the cellular ubiquitin ligase, E6-AP; however, it is presently not known how E6 targets the Drosophila discs large (Dlg) tumour suppressor and the membrane-associated guanylate kinase inverted (MAGI) family of proteins for degradation. By using an in vitro E6-AP immunodepletion assay, these targets were tested for degradation in a E6-AP-dependent manner. The data showed clearly that E6 can direct the degradation of Dlg and the MAGI family of proteins in the absence of E6-AP in this in vitro system. These results provide compelling evidence for the role of E6-associated ubiquitin ligases other than E6-AP in the degradation of certain E6 targets.

HPV E6 specifically targets different cellular pools of its PDZ domain-containing tumour suppressor substrates for proteasome-mediated degradation.

The high-risk HPV E6 proteins have been shown to direct the degradation of a variety of cellular proteins that contain PDZ domains. Although some of these proteins are involved in regulating processes of cell growth and polarity in Drosophila, little is known about their function in higher eukaryotic epithelial cells. In HPV-containing cells derived from cervical tumours, we find that the patterns of expression of the E6 targets hDlg (discs large), hScrib (Scribble), and MUPP1 are consistent with their being substrates for E6-induced degradation. It is also clear that, in the case of hDlg, E6 is specifically targeting nuclear pools of the protein rather than membrane-bound forms. We have also analysed the activity of a subset of E6 target proteins in the suppression of oncogene-induced cell transformation. Interestingly, Dlg, MAGI-1 and MUPP1 efficiently suppressed cell transformation, while MAGI-2 and MAGI-3 were ineffective in this assay. These results suggest that in the context of HPV-induced transformation Dlg, MAGI-1 and MUPP1 can function as tumour suppressors.

Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18.

Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types.