Dissection of the C-terminal region of E1A redefines the roles of CtBP and other cellular targets in oncogenic transformation.

Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.

The cell polarity regulator hScrib controls ERK activation through a KIM site-dependent interaction.

The cell polarity regulator, human Scribble (hScrib), is a potential tumour suppressor whose loss is a frequent event in late-stage cancer development. Little is yet known about the mode of action of hScrib, although recent reports suggest its role in the regulation of cell signalling. In this study we show that hScrib is a direct regulator of extracellular signal-regulated kinase (ERK). In human keratinocytes, loss of hScrib results in elevated phospho-ERK levels and concomitant increased nuclear translocation of phospho-ERK. We also show that hScrib interacts with ERK through two well-conserved kinase interaction motif (KIM) docking sites, both of which are also required for ERK-induced phosphorylation of hScrib on two distinct residues. Although wild-type hScrib can downregulate activation of ERK and oncogenic Ras co-transforming activity, an hScrib mutant that lacks the carboxy terminal KIM docking site has no such effects. These results provide a clear mechanistic explanation of how hScrib can regulate ERK signalling and begin to explain how loss of hScrib during cancer development can contribute to disease progression.

Human and primate tumour viruses use PDZ binding as an evolutionarily conserved mechanism of targeting cell polarity regulators.

A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways.

Human papillomaviruses, cervical cancer and cell polarity.

Human papillomaviruses (HPVs) are the causative agents of a number of human cancers, of which cervical cancer is the most important. This occurs following persistent infection with a limited number of viral subtypes and is characterized by continued expression of the viral E6 and E7 oncoproteins. A unique characteristic of the cancer-causing HPV types is the presence of a PDZ recognition motif on the carboxy terminus of the E6 oncoprotein. Through this motif, E6 directs the proteasome-mediated degradation of cellular proteins involved in the regulation of cell polarity and in cell proliferation control. These include components of the Scrib and Par polarity complexes, as well as a number of other PDZ domain-containing substrates. Thus, PVs are now providing novel insights into the functioning of many of these cellular proteins, and into which of these functions, in particular, are relevant for maintaining normal cellular homeostasis. In this review, we discuss the biological consequences of papillomaviral targeting of these cell polarity regulators, both with respect to the viral life cycle and, most importantly, to the development of HPV-induced malignancy.

HPV E6 degradation of p53 and PDZ containing substrates in an E6AP null background.

Human papillomavirus (HPV) type 16 and 18 E6 proteins target many of their cellular substrates for proteasome-mediated degradation. In the case of p53, this is mediated by the E6AP ubiquitin ligase. However it is still unclear whether other E6 substrates, in particular those containing PDZ domains, are also degraded in a similar manner. To investigate this, we established an epithelial cell line from E6AP-null mice and used these cells as a background to perform E6-mediated in vivo degradation assays. We show that the PDZ domain-containing substrates of E6, including Scribble, MAGI-1 and MAGI-3, are all subject to E6-mediated degradation in these cells. Strikingly, we also found that p53 could be degraded by E6 within these cells in a proteasome-dependent manner. These results demonstrate that HPV-16 and -18 E6 can target substrates for degradation in a manner independent of the E6AP ubiquitin ligase.

Phosphorylation of the discs large tumour suppressor protein controls its membrane localisation and enhances its susceptibility to HPV E6-induced degradation.

The Discs Large (Dlg) protein is intimately involved in regulating cell polarity and cell proliferation, and is targeted by the high-risk Human Papillomavirus (HPV) E6 proteins for proteasome-mediated degradation. We show here that exposure of cells to osmotic shock induces the hyperphosphorylation of Dlg and its concomitant accumulation within the cell membrane at sites of cell contact, a process that requires an intact actin filament network. In addition, hyperphosphorylation of Dlg also renders it more susceptible to degradation induced by the HPV-18 E6 oncoprotein. Mutational analysis of Dlg identifies a region within the first 185 amino acids as being important for this, with phosphorylation on residue S158 being responsible for its enhanced targeting by the E6 oncoprotein. Using specific kinase inhibitors, we show that Jun N-terminal kinase (JNK) is in part responsible for this phosphorylation, and for the subsequent Dlg accumulation at sites of cell contact. These results further support the notion of a complex phosphorylation-dependent regulation of Dlg, both with respect to its precise cellular localisation and to its susceptibility to proteasome-mediated degradation.

Proteasome-mediated regulation of the hDlg tumour suppressor protein.

The Dlg tumour suppressor protein is intimately involved in the control of cell contact and polarity. Previous studies have shown that hDlg is a target for a number of viral transforming proteins. In particular, the high risk human papillomavirus (HPV) E6 proteins target hDlg for proteasome-mediated degradation, an activity that appears to contribute to HPV-induced malignancy. However, little information is available concerning the normal regulation of hDlg. In this study we have investigated the role of the proteasome in the regulation of endogenous hDlg protein levels in epithelial cell lines. We demonstrate that hDlg is, indeed, degraded via the proteasome both in the presence and absence of HPV, in a fashion that is dependent on the ability of the cells to form cell junctions. By western blot and immunofluorescence analysis we show that hDlg is efficiently degraded in isolated cells; however, upon cell-cell contact, hDlg is both hyper-phosphorylated and stabilised. Strikingly, in both transformed rodent cells and undifferentiated cervical cancer cells, this ability to stabilise Dlg upon increased cell density is lost. These results demonstrate a complex pattern of hDlg regulation by phosphorylation and proteasome degradation in response to cell contact. Loss of this regulation probably represents a significant step in the development of malignancy.

Regulation of the human papillomavirus oncoproteins by differential phosphorylation.

Human papillomaviruses (HPVs) are intimately associated with the development of cervical cancer. The virus encodes two oncoproteins, E6 and E7, that are primarily responsible for inducing malignant transformation. The last few years have seen significant progress in elucidating the mechanisms by which these two viral proteins bring about cell transformation. Both proteins interact with a large number of cellular targets, many of which are involved in regulating diverse functions such as cell cycle regulation, transcription, differentiation and apoptosis. However both E6 and E7 are normally present at low levels within the virally infected cell, and how all these interactions are achieved and regulated has, until recently, been unclear. We have found that both E6 and E7 are subject to differential phosphorylation, the net results of which regulate their abilities to interact with some of their respective target proteins. In the case of E6, phosphorylation by Protein Kinase A (PKA) negatively regulates its ability to interact with the Discs Large (Dlg) tumour suppressor. In the case of E7, phosphorylation by Casein Kinase II (CKII) significantly increases its ability to interact with the TATA Box Binding Protein (TBP). Further, CKII regulation of E7 appears to vary during the cell cycle, therefore this provides a means of specifically targeting E7 to a given substrate at a given point within the cell cycle. This differential regulation of E6 and E7 by phosphorylation thus provides specificity to a diverse set of protein-protein interactions.

Differential phosphorylation of the HPV-16 E7 oncoprotein during the cell cycle.

The human papillomavirus type 16 encodes two principal oncoproteins, E6 and E7. The E7 protein has been shown to deregulate the cell cycle through interactions with a variety of proteins involved in cell cycle control and transcriptional regulation. These activities result in E7 being able to cooperate with activated oncogenes in the transformation of primary rodent cells, and with the viral E6 protein during human keratinocyte immortalization. Although a large number of activities have been ascribed to the E7 protein, little is known about its regulation during the cell cycle. We have performed a series of studies to investigate potential changes in E7 phosphorylation during the cell cycle and we show that E7 is indeed differentially phosphorylated. Casein kinase II is the principal kinase during the early part of the cell cycle, but this activity decreases rapidly as cells progress toward S phase. In addition, E7 is transiently phosphorylated at Ser71 in S phase by another, as yet unknown, kinase. These results demonstrate differential regulation of the E7 protein during the cell cycle that most likely represents a means of providing specificity to E7's activities.

Adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 E2 recruitment of the transcriptional coactivator p300.

Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300.

Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1.

We had previously observed that HPV-16 E7 disturbs the Guanylate Binding Protein (GBP)-ISRE reporter activation by IFN-gamma thus suggesting an alteration of the IRF-1 function. In this study we examined the mechanism by which E7 affects the IFN-gamma signals driving the activation of gene transcription. Using 14/2 BRK cells containing dexamethasone-inducible HPV-16 E7 gene, we observed a large inhibition of the IRF-1 DNA binding activity upon E7 induction. Concomitantly, there was no significant change in the levels of IRF-1, indicating that this was not due to reduced levels of IRF-1 expression. Likewise, in vitro translated E7 did not affect the IRF-1 DNA binding activity in nuclear extracts derived from IFN-induced cells, thus indicating that the effects of E7 are upstream of IRF-1's binding to its DNA recognition site. Finally, NFkappaB DNA binding activity was also inhibited under conditional expression of E7. These data indicate that HPV-16 E7 inhibits the IRF-1 and NFkappaB function and this could lead to the impairment of the IFN response in HPV-infected cells. Furthermore, the findings suggest that different events of the IFN inducible signal cascade seem to be target for HPV-16 E7.

Allosteric activation of acid alpha-glucosidase by the human papillomavirus E7 protein.

Changes in the cellular carbohydrate metabolism are a hallmark of malignant transformation and represent one of the earliest discernible events in tumorigenesis. In the early stages of certain epithelial cancers, a metabolic switch is regularly observed, in which slowly growing glycogenotic cells are converted to highly proliferating basophilic cells. This step is accompanied by a rapid depletion of the intracellular glycogen stores, which in liver carcinogenesis results from the activation of the enzyme acid alpha-glucosidase by an as yet unknown mechanism. We show here that acid alpha-glucosidase is a target for the E7 protein encoded by human papillomavirus type 16, a human tumor virus that plays a key role in the genesis of cervical carcinoma. We show that expression of E7 induces the catalytic activity of acid alpha-glucosidase in vivo and wild type E7, but not transformation-deficient mutants bind directly to acid alpha-glucosidase and increase the catalytic activity of the enzyme in vitro. The data suggest that the E7 protein encoded by human papillomavirus type 16 can act as an allosteric activator of acid alpha-glucosidase.

Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein.

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.

Interaction between the HPV-16 E2 transcriptional activator and p53.

The HPV-16 E2 protein is a major regulator of viral DNA replication and gene expression. Through interactions with the viral origin binding protein, E1, it localizes E1 to the origin of replication and stimulates the initiation of viral DNA replication. However, several recent reports have described a number of diverse activities of E2 relating to the induction of apoptosis through both p53 dependent and independent mechanisms, and to induction of growth arrest in both the G1 and G2M phases of the cell cycle. Recent studies have also shown that p53 can specifically inhibit HPV DNA replication, albeit through an unknown mechanism. Since p53 has been described in the replication centres of Herpes Viruses, Adenovirus and SV40 we decided to investigate whether any of the above activities of E2 may be related to an association with p53. We show, in a series of in vitro assays, specific interaction between p53 and HPV-16 E2 via residues in the carboxy terminal half of the E2 protein. Mutational analysis of p53 indicates that sequences in both the DNA binding and oligomerization domains are essential for the interaction, and a mutant of p53 which is unable to bind E2 is also unable to inhibit HPV DNA replication. Finally, using an inducible system of p53 expression we also show that E2 will complex with p53 in vivo. These results raise the intriguing possibility that p53 may also be involved in HPV DNA replication centres, and also provides explanations for some of the diverse activities reported for the HPV E2 proteins.

Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth.

The E6 proteins originating from the tumour-associated Human Papillomavirus (HPV) types 16 and 18 have been shown to bind to and target the tumour suppressor protein, p53, for ubiquitin-mediated degradation. However, in cell lines derived from cervical neoplasias, the predominant early region transcripts are spliced and encode truncated forms of E6, termed E6*. We report here that HPV-18 E6* protein will interact both with the full-length E6 proteins from HPV-16 and HPV-18 and also with E6-AP, and subsequently blocks the association of full length E6 protein with p53. We also show that, as a result of this block, E6* can inhibit E6-mediated degradation of p53 both in vitro and in vivo. The biological consequences of this are increased transcriptional activity on p53-responsive promoters and an inhibition of cell growth in cells transfected with E6*. This is the first report of a potential biological function for this polypeptide and may represent a means by which HPV is able to modulate the activity of the full-length E6 protein with respect to p53 during viral infection.

Repression of p53 transcriptional activity by the HPV E7 proteins.

The major transforming protein of human papillomaviruses (HPVs) is encoded by the E7 gene. This protein cooperates with activated oncogenes to transform primary rodent cells and with the viral E6 gene to immortalize primary human keratinocytes. Numerous cellular targets of HPV E7 have now been identified including pRb, p107, cyclin A, TATA box binding protein (TBP), and members of the AP-1 transcription factor family. As with Adenovirus E1a, many of these interactions are important for the ability of E7 to transform cells. Recent studies have demonstrated that Adenovirus E1a can also inhibit the transcriptional activity of the cellular tumor suppressor protein, p53. We have performed a series of analyses to determine whether HPV E7 proteins share this characteristic. We show that HPV E7 proteins derived from both benign and tumor-associated HPV types are able to inhibit p53 transcriptional activity. Mutational analysis of the HPV-16 E7 protein reveals that a key domain involved in mediating this activity is the casein kinase II (CKII) recognition site, which has been shown to modulate E7 binding to TBP. We further show that E7 does not bind to p53 directly, but will do so in the presence of exogenously added TBP and that this binding is increased following CKII phosphorylation. These results suggest that the E7-TBP interaction may be responsible for inhibiting p53 transcriptional activity.

Human papillomavirus type 16 E7 binds to the conserved carboxy-terminal region of the TATA box binding protein and this contributes to E7 transforming activity.

We have previously shown that the human papillomavirus E7 proteins bind to the cellular TATA box binding protein (TBP). In this paper we show that the HPV-18 E6 and the HPV-16 E2 proteins will also bind TBP in vitro. This feature of virus proteins is conserved across many viral types and we were interested in determining whether these HPV proteins interacted with the same conserved region of the TBP molecule. A series of deletions was introduced into the TBP protein and its binding to these HPV proteins was measured. The previously well-characterized interaction between p53 and TBP was used for comparison. All four proteins were found to interact with the carboxy-terminal domain of the TBP protein, although the precise residues involved and the relative strengths of association differed between the different HPV proteins. Mutational analysis of HPV-16 E7 protein identified a stretch of four amino acids responsible for the binding to TBP. This mutant E7 protein possessed wild-type levels of transcriptional activity on the adenovirus E2 promoter but exhibited reduced transforming activity in cooperation with EJ-ras. These results demonstrate that the mechanisms of interaction between diverse viral proteins and TBP are similar and that, in the case of E7, this interaction may contribute to its transforming activity.

HPV-18 E6 inhibits p53 DNA binding activity regardless of the oligomeric state of p53 or the exact p53 recognition sequence.

The E6 proteins of the oncogenic-associated human papillomavirus types 16 (HPV-16) and 18 (HPV-18) function by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. HPV E6 is able to interfere with p53 function by preventing its binding to DNA target sequences and also by labelling p53 for ubiquitin-mediated degradation. We have previously reported that certain p53 mutants, defective in oligomerisation, vary in their susceptibility to E6-directed labelling for ubiquitin-mediated degradation. In this paper we report that the strength of p53's binding to DNA is dependent upon the precise target sequence, but that E6 is able to disrupt each complex. We also report the binding of different oligomeric forms of p53 to different DNA sequences and correlate this with in vivo transcriptional activity and demonstrate the susceptibility of that DNA binding to disruption by E6. Finally we show that the ability of p53 to bind to TBP is a function of its oligomeric state and correlates in part with its ability to transrepress but not with its ability to transactivate.

HPV-16 E7 and adenovirus E1a complex formation with TATA box binding protein is enhanced by casein kinase II phosphorylation.

The major transforming protein of HPV-16 is encoded by the E7 gene. This has been shown to cooperate with EJ-ras in the immortalisation of primary rodent cells and with the viral E6 gene in the immortalisation of primary human keratinocytes. HPV-16 E7 protein has been shown to bind to a number of cellular proteins involved in the control of cell growth; including pRB, p107 and cyclin A. Loss of pRb or p107 binding results in the loss of transforming activity. In this paper we demonstrate that HPV-16 E7 can also complex with the core component of TFIID, the TATA Box Binding Protein (TBP). This interaction is partly dependent upon phosphorylation of the E7 protein by cellular casein kinase II (CKII), since phosphorylation of E7 by CKII increases the affinity with which E7 binds TBP. Similar results are also obtained with the Adenovirus Ela protein, indicating a conservation of function between these two viral oncoproteins. Mutation of the CKII site to two acidic amino acids significantly increases the affinity of E7 for TBP, indicating that the incorporation of two negative charges at this region of E7 is important in regulating the interaction with TBP.

Characterisation of cellular changes which influence progression of human papillomavirus type-16 immortalised keratinocytes to anchorage-independent phenotype.

Cellular alterations which influence progression to an anchorage-independent phenotype are poorly understood. Few immortalised keratinocyte lines have been reported to form colonies in semi-solid medium, and the important role of the anchorage-dependence in controlling the behaviour of keratinocytes reflects the resistance of these cells to form colonies in soft agar. We describe here a model for studying in vitro the progression of human papillomavirus type-16 (HPV-16) immortalised keratinocytes from the early stages of immortalisation to an anchorage-independent phenotype. By an extensive selection procedure we have isolated three related cell lines, one immortalised, one weakly anchorage independent and one completely anchorage-independent. The comparison of these three lines gave a clear indication that this in vitro altered growth property is mainly correlated with a deregulation of the epidermal growth factor receptor (ECFR), leading to an increased proliferation rate of the cells coupled with changes in keratin expression.