Biomedical subjects

L Banks

Publications and source records attributed to L Banks.

At least 19 recordsLinked to original sources

PubMed2006

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.

Adaptor Proteins, Signal Transducing↗

PubMed2001

The human papillomavirus E6 protein and its contribution to malignant progression.

The human papillomavirus (HPV) E6 protein is one of three oncoproteins encoded by the virus. It has long been recognized as a potent oncogene and is intimately associated with the events that result in the malignant conversion of virally infected cells. In order to understand the mechanisms by which E6 contributes to the development of human malignancy many laboratories have focused their attention on identifying the cellular proteins with which E6 interacts. In this review we discuss these interactions in the light of their respective contributions to the malignant progression of HPV transformed cells.

Cell Transformation, Neoplastic↗

PubMed2001

The HPV-16 E7 oncoprotein binds Skip and suppresses its transcriptional activity.

E7 is the major transforming protein of human papillomavirus (HPV), which is implicated in the development of cervical cancer. The transforming activity of E7 has been attributed in part to its interaction with the retinoblastoma (Rb) tumour suppressor; however, the Rb interaction alone is not sufficient for transformation by E7. In a screen for cellular targets of HPV E7, we identified the Ski interacting protein, Skip, as a new interacting partner of E7. We show that HPV-16 E7 associates with Skip via sequences in its carboxy terminal region, and the evolutionarily conserved proline rich sequences (PRS) of the SNW domain of Skip. E7 functionally targets Skip in vivo and inhibits its transcriptional activation activity. Two transformation defective mutants of E7 were identified that failed both to bind Skip and to inhibit its transcriptional activity. These results suggest that inhibition of Skip function may contribute to cell transformation by HPV-16 E7.

Binding Sites↗

PubMed2001

HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation.

It has recently been shown that the high-risk human papillomavirus (HPV) E6 proteins can target the PDZ-domain containing proteins, Dlg, MUPP-1, MAGI-1 and hScrib for proteasome-mediated degradation. However, the E6 proteins from HPV-16 and HPV-18 (the two most common high-risk virus types) differ in their ability to target these proteins in a manner that correlates with their malignant potential. To investigate the underlying mechanisms for this, we have mutated HPV-16 and HPV-18 E6s to give each protein the other's PDZ-binding motif. Analysis of these mutants shows that the greater ability of HPV-18 E6 to bind to these proteins and to target them for degradation is indeed due to a single amino acid difference. Using a number of assays, we show that the E6 proteins interact specifically with only one of the five PDZ domains of MAGI-1, and this is the first interaction described for this particular PDZ domain. We also show that the guanylate kinase homology domain and the regions of MAGI-1 downstream of amino acid 733 are not required for the degradation of MAGI-1. Finally, in a series of comparative analyses, we show that the degradation of MAGI-1 occurs through a different mechanism from that used by the E6 protein to induce the degradation of Dlg and p53.

Amino Acid Motifs↗

PubMed2001

Ski interacts with the evolutionarily conserved SNW domain of Skip.

Ski interacting protein (Skip) has been found to bind to the highly conserved region of Ski, which is required for its transforming activity. Ski is a unique oncoprotein that is involved in inducing both transformation and differentiation. At the molecular level, Ski has been shown to exhibit either co-activator or co-repressor activity depending on the cellular and promoter context. We were interested in further elucidating the biological implications of the Ski-Skip interaction. Here we have identified the SNW domain of Skip as the interaction region for Ski. This domain of Skip is highly conserved in all the Skip homologues identified from different species. Using a series of reporter plasmids, we show that Skip is a potent transcriptional activator of many different promoters, the activity of which was also mapped to the conserved core SNW domain of the protein. Addition of excess Ski further augmented the transcriptional activities of Skip, suggesting that one of the ways in which Ski brings about transformation is by binding and cooperating with the SNW domain of Skip in transcriptional activation.

Binding Sites↗

PubMed2001

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.

Adaptor Proteins, Signal Transducing↗

PubMed2001

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.

Animals↗

PubMed2000

Role of Bak in UV-induced apoptosis in skin cancer and abrogation by HPV E6 proteins.

Ultraviolet B (UVB) damage is recognized as the most important etiological factor in the development of skin cancer. Human papillomaviruses (HPV) have also been implicated in the disease, although the mechanism of action of these viruses remains unknown. We present evidence here that Bak protein is involved in signaling apoptosis in the skin in response to UVB damage, and that cutaneous HPV E6 proteins target and abrogate Bak function by promoting its proteolytic degradation both in vitro and in regenerated epithelium. Additionally, HPV positive skin cancers had undetectable levels of Bak in contrast to HPV negative cancers, which expressed Bak. This study supports a link between the virus and UVB in the induction of HPV-associated skin cancer and reveals a survival mechanism of virally infected cells.

Apoptosis↗

PubMed2000

Differential regulation of human papillomavirus E6 by protein kinase A: conditional degradation of human discs large protein by oncogenic E6.

The protein Kinase A (PKA) pathway was found to selectively regulate the function of oncogenic but not non-oncogenic E6 proteins. High risk E6 proteins are phosphorylated at their Dlg/PDZ binding motif at the C-terminus by a PKA like activity. This PKA and PDZ binding module is found only for human PV, is strictly conserved in all the transforming HPVs and is absent in all the low risk HPV types. We present evidence of a conditional regulation of E6 induced degradation of Dlg. HPV18E6 positive but not HPV negative keratinocytes exhibit increased Dlg steady state levels under conditions of high PKA activity, with a concomitant increase in the presence of Dlg at tight junctions. In vitro binding experiments show that E6 phosphorylation by PKA reduces its binding to Dlg and molecular modelling can explain this observation in a structural context. E6 dependent degradation of Dlg in cells with high PKA levels is inhibited and this is dependent on phosphorylation of the PDZ binding site in E6. In contrast, the degradation of p53 induced by E6 is not affected by PKA. We propose a differential regulation of E6 for the ubiquitin mediated degradation of specific E6 target proteins.

3T3 Cells↗

PubMed2000

Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins.

The oncoproteins of small DNA tumor viruses promote tumorigenesis by complexing with cellular factors intimately involved in the control of cell proliferation. The major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 proteins, respectively. These seemingly unrelated viral oncoproteins are similar in that their transforming activities in cells depend, in part, on a carboxyl-terminal PDZ domain-binding motif which mediates interactions with the cellular PDZ-protein DLG. Here we demonstrated that both Ad9 E4-ORF1 and high-risk HPV E6 proteins also bind to the DLG-related PDZ-protein MAGI-1. These interactions resulted in MAGI-1 being aberrantly sequestered in the cytoplasm by the Ad9 E4-ORF1 protein or being targeted for degradation by high-risk HPV E6 proteins. Transformation-defective mutant viral proteins, however, were deficient for these activities. Our findings indicate that MAGI-1 is a member of a select group of cellular PDZ proteins targeted by both adenovirus E4-ORF1 and high-risk HPV E6 proteins and, in addition, suggest that the tumorigenic potentials of these viral oncoproteins depend, in part, on an ability to inhibit the function of MAGI-1 in cells.

Adenoviridae↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Allosteric Regulation↗

PubMed2000

HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase.

The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We first show that the differences in the affinities of HPV-16 and of HPV-18 E6 proteins for binding DLG is reflected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to define regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Significant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most significantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

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.

DNA Helicases↗

PubMed2000

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

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

3T3 Cells↗

PubMed2000

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.

Cell Line↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

The role of the E6-p53 interaction in the molecular pathogenesis of HPV.

Human papillomaviruses (HPVs) are associated with a number of clinical conditions, of which the most serious is cervical carcinoma. The E6 protein of the oncogenic, mucosal-specific HPV types has been shown to complex with p53 and, as a result, target it for rapid proteasome-mediated degradation. As a consequence, p53's growth-arrest and apoptosis-inducing activities are abrogated. Since p53 is frequently wild type in cervical cancers, unlike other cancers in which it is often mutated, the notion has arisen that E6's activity with respect to p53 is equivalent to an inactivating mutation of p53. In addition, several studies have shown that the pathways both upstream and downstream of p53 are intact in cervical cancers; this suggests the potential importance of the E6 - p53 interaction for therapeutic intervention. However, like all viral oncoproteins, E6 is a multifunctional protein and a plethora of other cellular targets has been identified. Indeed, E6's interactions with some of these additional targets appear to be equally important in the pathogenesis of HPV, and may also represent valid targets for therapeutic intervention.

Alternative Splicing↗