Biomedical subjects

Lawrence Banks

Publications and source records attributed to Lawrence Banks.

32 records · Page 2Linked to original sources

PubMed2003

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint.

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3'-hydroxyl and 5'-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.

Animals↗

PubMed2003

Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes.

Human papillomavirus E6 oncoproteins induce the proteasomal degradation of several multi-PDZ (PSD95/Dlg/ZO-1) domain-containing proteins such as the human homologue of Drosophila discs large. Binding to PDZ domain-containing proteins is mediated by a PDZ-binding motif contained within the C-terminus of E6. The ability of E6 proteins to induce degradation of PDZ domain-containing proteins correlates with their oncogenic potential. Here we examined the biological effect of this region of the human papillomavirus type 18 E6 oncoprotein on keratinocyte morphology. Our results show that in simian virus 40-immortalized human keratinocytes, stable expression of E6 correlated with the induction of an exaggerated mesenchymal-like morphology and actin cytoskeleton disorganization compared with parental cells. The altered phenotype was accentuated in cells expressing an E6 protein containing a mutation (Arg153Leu) within a protein kinase A recognition motif that abrogates protein kinase A's negative regulation of the activity of the PDZ-binding domain. The E6-induced changes indicated an epithelial-mesenchymal transition and were supported by the finding that E6-expressing cells contained vimentin. Changes to the epithelial phenotype of cells expressing a mutant E6 protein (Thr156Glu) that is unable to degrade discs large was significantly less marked, although they did show evidence of epithelial-mesenchymal transition. These observations imply that the activity of the E6 PDZ-binding motif contributes only to a part of the transition. Further analysis of the E6 cell lines showed a decrease in adherens junction and desmosome formation. Cells expressing a functional PDZ-binding motif showed the greatest disruption of intercellular junction formation, but this did not correlate with a decrease in total cellular levels of the individual components of adhesion junctions. This suggests that the activity of the PDZ-binding motif may have influenced either the assembly or integrity of functional adhesion complexes. An E6-mediated decrease in peripheral membrane levels of PDZ proteins like discs large could be the basis for the enhanced morphological transformation of immortalized keratinocytes.

Actins↗

PubMed2003

Redistribution of the discs large tumor suppressor protein during mitosis.

Drosophila discs large (Dlg) has been shown to be an essential regulator of cell polarity and attachment, and is classified as a potential tumour suppressor in higher eukaryotes. Human Dlg is expressed in epithelial cells at sites of cell-cell contact and acts as a negative regulator of cell growth. Although hDlg has been shown to be phosphorylated during mitosis, little is known about its activity during this stage of the cell cycle. To investigate this further we have analysed in detail the pattern of hDlg expression during mitotic cell division. In early mitosis there is a marked increase in membrane-bound hDlg which is then retained throughout mitosis, while during cytokinesis, there is a specific concentration of hDlg at the midbody. Using mutants of Dlg we show that this is mediated by sequences in the carboxy terminal region of Dlg, but it does not require the SH3 or PDZ domains, and is independent of binding to protein 4.1. Finally, using a mutant of Dlg that consists of just this carboxy terminal region of the protein, we show that it can compete with endogenous hDlg for midbody accumulation, and this mutant also gives rise to altered cell growth. We conclude that localisation of Dlg to the midbody indicates a role for Dlg at this critical point in cytokinesis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300.

Infection with high-risk human papillomaviruses (HPV) can lead to the development of cervical cancer. This process depends on the interaction of the virus-encoded oncoproteins, E6 and E7, with a variety of host regulatory proteins. As E7 shares both functional and structural similarities with the Adenovirus E1a (Ad E1a) protein, we were interested in investigating the possible interactions between E7 and the transcriptional coactivator p300, since it was originally identified as a target of Ad E1a. Using a variety of assays, we show that E7s from both high- and low-risk HPV types interact with p300. Mutational analysis of E7 maps the site of the interaction to a region spanning the pRb-binding domain and the CKII phosphorylation site. We also map the site of interaction on p300 largely to the CH1 domain. In addition, we demonstrate that the binding between 16E7 and p300 is direct, and can be detected in vivo by coimmunoprecipitation and mammalian two-hybrid assays. Finally, we show that E7 can abolish the p300-mediated E2 transactivation function, suggesting that complex formation between E7 and p300 may contribute to the regulation of E2 transcriptional activity.

Animals↗

PubMed2003

Regulation of the discs large tumor suppressor by a phosphorylation-dependent interaction with the beta-TrCP ubiquitin ligase receptor.

The discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Viruses and the 26S proteasome: hacking into destruction.

The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus-host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases.

Adenoviridae↗

PubMed2002

Skip interacts with the retinoblastoma tumor suppressor and inhibits its transcriptional repression activity.

Ski interacting protein (Skip) plays an important role in the transforming activity of both v-Ski and EBNA2 (Epstein-Barr virus encoded latency protein) and is involved in EBNA2 and NotchIC activation of CBF1-repressed promoters. We have previously shown that Skip acts as a transcriptional co-activator on a number of cellular and viral promoters. Here, we report that Skip also interacts with pRb and, in cooperation with Ski, can overcome pRb-induced transcriptional repression. We show a strong and direct interaction between pRb and Skip, and we map the site of interaction to amino acid residues 171-353 of the evolutionarily conserved SNW domain of Skip. Furthermore, the combination of Skip and Ski can successfully overcome the G1 arrest and flat cell phenotype induced by pRb. Taken together, these studies suggest that one potential function of the Skip-Ski complex is to overcome the growth-suppressive activities of pRb.

Animals↗

PubMed2002

Chimaeric HPV E6 proteins allow dissection of the proteolytic pathways regulating different E6 cellular target proteins.

The ability of HPV E6 oncoproteins to induce the degradation of PDZ domain-containing MAGUK proteins correlates with their malignant potential. We previously showed that the HPV-6 E6 protein, when provided with the PDZ-binding domain from HPV-18 E6, acquires the ability to bind the Discs Large (Dlg) tumour suppressor and target it for degradation. Based on this finding we have extended this analysis to E6 proteins from a variety of different papillomavirus types. Cloning a PDZ-binding sequence onto the C-terminus of E6 proteins derived from low-risk mucosal, and low and high-risk cutaneous papillomavirus types, enables them to bind Dlg and a second MAGUK family member, MAGI-1. This renders the mucosally-derived low-risk chimaeric HPV E6 proteins capable of targeting Dlg for degradation, but they are unable to induce significant levels of degradation of MAGI-1. In contrast, none of the E6 proteins derived from cutaneous papillomavirus types induce significant degradation of either MAGI-1 or Dlg when provided with a PDZ-binding domain. These results demonstrate significant differences, both between mucosal and cutaneous HPV E6 proteins and in the pathways required for Dlg and MAGI-1 degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation.

The E6 proteins from the high-risk human papillomavirus (HPV) types have previously been shown to target a number of PDZ domain-containing proteins for proteasome-mediated degradation. These include the hDlg tumour suppressor and the MAGI-1 protein. In this study we show that high-risk HPV E6 proteins also target the related MAGI-2 and MAGI-3 proteins for degradation. Moreover, we show that the interaction is specific to one PDZ domain, and that co-expression of this domain can protect each of the full-length MAGI proteins from E6-mediated degradation. These data provide clear indicators for the potential design of compounds that could specifically inhibit the interaction of oncogenic HPV E6 proteins with an important class of target proteins.

Activin Receptors, Type II↗

PubMed2002

Comparative analysis of the intracellular location of the high- and low-risk human papillomavirus oncoproteins.

We have compared the intracellular location of the HPV E6 and E7 proteins from high- and low-risk virus types. While high-risk HPV E7 displays diffuse nuclear expression, low-risk E7 has a nuclear punctuate pattern of expression. Similarly, while high-risk E6 is expressed throughout the cell, low-risk E6 is again predominantly nuclear with a punctuate pattern of expression. Both low-risk viral oncoproteins show colocalization with PML, whereas high-risk viral proteins do not. Finally, inhibition of the proteasome pathway results in a dramatic nuclear accumulation of high-risk E6 protein. These results demonstrate fundamental differences in the localization of these viral oncoproteins within the cell and offer alternative explanations for their respective differences in pathology.

DNA-Binding Proteins↗

PubMed2002

Human papillomavirus-16 E7 protein inhibits the DNA interaction of the TATA binding transcription factor.

Previous studies have shown that the HPV-16 E7 protein interacts with TBP. This interaction was found to take place through residues in the carboxy terminal half of E7, mutation of which resulted in weaker transforming activity. In addition, binding of E7 to TBP was found to be increased following protein kinase CK2 (casein kinase II) phosphorylation of E7, and mutation of this CK2 site also reduces E7's transforming activity. To date, however, there is no information on the effects of E7 upon TBP function. In order to address this we have performed a series of assays to investigate the effects of E7 upon the ability of human and S. pombe TBP to bind DNA. We show that HPV-16 E7 is indeed a potent inhibitor of TBP DNA binding activity. Further, this activity of E7 is increased following CK2 phosphorylation of E7, consistent with it having an increased affinity for TBP. Finally, a mutant E7 protein defective in its ability to bind TBP, has no effect upon TBP binding to DNA. These results demonstrate that one consequence of the E7-TBP interaction is abolition of TBP DNA binding activity, and may provide an explanation for the transcriptional inhibitory effects of E7.

Animals↗

PubMed2002

Changes in expression of the human homologue of the Drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias.

The Drosophila tumour suppressor discs large (Dlg) is a cell-junction localized protein that is required for the maintenance of epithelial cyto-architecture and the negative control of cell proliferation. The mammalian homologue is likely to have a similar mode of action, and therefore functional perturbation of this protein may be linked to the development of epithelial-derived cancers. The finding that several unrelated viral oncoproteins, including the E6 protein of oncogenic human papillomaviruses, bind to the human homologue of Dlg (hDlg) supports this proposition. Employing immunohistochemistry, we show that in uterine cervical squamous epithelia, prominent localization of hDlg at sites of intercellular contact occurs in cells that have left the proliferating basal cell layers and begun maturation. The presence of hDlg at sites of cell:cell contact diminishes, whilst intracellular cytoplasmic levels increase significantly in high-grade, but not low-grade, cervical neoplasias. In invasive squamous cell carcinomas, total cellular hDlg levels are greatly reduced. Our data suggest that loss of hDlg at sites of intercellular contact may be an important step in the development of epithelial cancers.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Mutational analysis of the discs large tumour suppressor identifies domains responsible for human papillomavirus type 18 E6-mediated degradation.

The discs large (Dlg) tumour suppressor protein is targeted for ubiquitin-mediated degradation by the high-risk human papillomavirus E6 proteins. To understand further the mechanisms behind this, a mutational analysis of Dlg was undertaken. This study demonstrates that an intact PDZ domain 2 (PDZ2) on Dlg is necessary for the ability of E6 to bind and degrade Dlg. However, additional residues within the amino-terminal portion of Dlg are also required for optimal E6 activity. Stable cell lines expressing different Dlg mutants were also established and these confirm that Dlg is regulated intrinsically by the proteasome in the absence of E6; however, in this case, the sequences responsible for regulating Dlg stability lie predominantly within PDZ2. These results suggest that there are at least two mechanisms for regulating Dlg protein stability and that the pathways used by E6 are not necessarily the same as those used in the cell in its absence.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Complementation of a p300/CBP defective-binding mutant of adenovirus E1a by human papillomavirus E6 proteins.

Previous studies have shown that the human papillomavirus type 16 (HPV-16) E6 protein binds to p300/CBP and abrogates its transcriptional co-activator function. However, there is little information on the biological consequences of this interaction and discrepancy as to whether the interaction is high-risk E6 specific or not. We performed a series of studies to compare the interactions of HPV-18 and HPV-11 E6 with p300, and showed that both high- and low- risk E6 proteins bind p300. In addition, using a transformation-deficient mutant of adenovirus E1a, which cannot interact with p300, we demonstrated that HPV-16, HPV-18 and, to a lesser extent, HPV-11 E6, can complement this mutant in cell transformation assays. In contrast, a mutant of HPV-16 E6 which does not bind p300 failed to rescue the E1a mutant. These results suggest that the E6-p300 interaction may be important for the ability of HPV E6 to contribute towards cell transformation.

Adenovirus E1A Proteins↗