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The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

Krishna Subbaiah V, Massimi P, Boon SS, Myers MP, Sharek L, Garcia-Mata R, Banks L - PLoS Pathog. (2012)

Bottom Line: We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity.In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6.Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

ABSTRACT
A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

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High levels of RhoG activity in HPV-18 transformed cells is hDlg and HPV -dependent.Panel A. HeLa cells were transfected with siRNAs against luciferase (Luc) control, E6/E7, hDlg or SGEF as indicated. After 72 hrs cell extracts were then incubated with purified GST.ELMO to determine the levels of active RhoG, which was detected by western blotting (upper panel). The lower panels show input levels of total RhoG, hDlg, SGEF, p53 and α-Actinin. Also shown is the Ponceau stain of the membrane showing constant levels of GST.ELMO. The graph shows the quantifications from multiple GST.ELMO pull-downs, and shows the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent +/− SD of four independent experiments. Note the modest decrease in active RhoG following the removal of E6/E7 and the dramatic decrease following removal of hDlg. Panel B. HeLa cells were transfected with control siRNA (Luc) siRNA to E6/E7 (i) or siRNA to hDlg-1 (ii) and were then analysed for the levels of hDlg, SGEF, and p53 expression after 72 hrs in the insoluble and soluble compartments of the cell. α-Actinin was used as a loading control. Note the marked increase in hDlg levels in the soluble fraction with concomitant decrease in the insoluble fraction following E6/E7 removal, which is also accompanied by a decrease in SGEF levels in this compartment and a similar loss of SGEF in the insoluble compartment is seen following siRNA ablation of hDlg-1 expression. Panel C. HeLa cells were transfected with control siRNA (Luc), siRNA to E6 (si18E6intronic) or siRNA to E6/E7 and were then analysed for levels of expression of SGEF and p53 in total cell lysates after 72 hrs. α-Actinin was used as a loading control.
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ppat-1002543-g006: High levels of RhoG activity in HPV-18 transformed cells is hDlg and HPV -dependent.Panel A. HeLa cells were transfected with siRNAs against luciferase (Luc) control, E6/E7, hDlg or SGEF as indicated. After 72 hrs cell extracts were then incubated with purified GST.ELMO to determine the levels of active RhoG, which was detected by western blotting (upper panel). The lower panels show input levels of total RhoG, hDlg, SGEF, p53 and α-Actinin. Also shown is the Ponceau stain of the membrane showing constant levels of GST.ELMO. The graph shows the quantifications from multiple GST.ELMO pull-downs, and shows the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent +/− SD of four independent experiments. Note the modest decrease in active RhoG following the removal of E6/E7 and the dramatic decrease following removal of hDlg. Panel B. HeLa cells were transfected with control siRNA (Luc) siRNA to E6/E7 (i) or siRNA to hDlg-1 (ii) and were then analysed for the levels of hDlg, SGEF, and p53 expression after 72 hrs in the insoluble and soluble compartments of the cell. α-Actinin was used as a loading control. Note the marked increase in hDlg levels in the soluble fraction with concomitant decrease in the insoluble fraction following E6/E7 removal, which is also accompanied by a decrease in SGEF levels in this compartment and a similar loss of SGEF in the insoluble compartment is seen following siRNA ablation of hDlg-1 expression. Panel C. HeLa cells were transfected with control siRNA (Luc), siRNA to E6 (si18E6intronic) or siRNA to E6/E7 and were then analysed for levels of expression of SGEF and p53 in total cell lysates after 72 hrs. α-Actinin was used as a loading control.

Mentions: Having shown that hDlg influences the activity of SGEF and RhoG, we wanted to determine what effects HPV-18 E6 might have upon this. To do this we first analysed the levels of RhoG activity in HPV-18 positive cervical tumour derived HeLa cells. GST-ELMO pull-downs were done using extracts from cells in which E6/E7 and hDlg1 expression had been ablated by siRNA transfection. The results and quantitations from multiple assays in Figure 6A show a number of interesting features. Firstly, RhoG activity levels are constitutively high in the control transfected cells. Ablation of E6/E7 expression results, as expected, in an increase in the total levels of hDlg expression and also, most surprisingly, results in a concomitant decrease in the levels of active RhoG. However, ablation of the residual hDlg alone, by siRNA transfection or in combination with siRNA E6/E7 also results in a dramatic decrease in the levels of active RhoG. These results suggest that high levels of RhoG activity in HeLa cells are dependent upon the presence of both E6/E7 and hDlg, even though certain pools of hDlg are also degraded by the HPV-18 E6 oncoprotein. This would therefore suggest that E6 is potentially recruiting or preserving a pool of hDlg that contributes to maintaining elevated SGEF activity. To investigate this possibility we analysed the levels of hDlg expression in the NP-40 soluble and insoluble pools of HeLa cells following ablation of E6/E7 expression. The results in Figure 6B(i) show that, as expected, there is a significant increase in the levels of hDlg expression in the NP-40 soluble fraction upon removal of E6//E7 expression. However, in the insoluble fraction there is a significant decrease in the levels of both hDlg and SGEF expression, suggesting that whilst certain pools of hDlg are targeted for degradation by E6, others are either actively maintained or unaffected. We also wanted to determine if the pattern of SGEF expression in other HPV positive cells was similarly dependent upon E6/E7 expression. To do this we analysed HPV-16 positive CaSki cells. The results in Figure S2 confirm that loss of E6/E7 expression also results in a loss of SGEF from the NP-40 insoluble pool in these cells.


The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

Krishna Subbaiah V, Massimi P, Boon SS, Myers MP, Sharek L, Garcia-Mata R, Banks L - PLoS Pathog. (2012)

High levels of RhoG activity in HPV-18 transformed cells is hDlg and HPV -dependent.Panel A. HeLa cells were transfected with siRNAs against luciferase (Luc) control, E6/E7, hDlg or SGEF as indicated. After 72 hrs cell extracts were then incubated with purified GST.ELMO to determine the levels of active RhoG, which was detected by western blotting (upper panel). The lower panels show input levels of total RhoG, hDlg, SGEF, p53 and α-Actinin. Also shown is the Ponceau stain of the membrane showing constant levels of GST.ELMO. The graph shows the quantifications from multiple GST.ELMO pull-downs, and shows the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent +/− SD of four independent experiments. Note the modest decrease in active RhoG following the removal of E6/E7 and the dramatic decrease following removal of hDlg. Panel B. HeLa cells were transfected with control siRNA (Luc) siRNA to E6/E7 (i) or siRNA to hDlg-1 (ii) and were then analysed for the levels of hDlg, SGEF, and p53 expression after 72 hrs in the insoluble and soluble compartments of the cell. α-Actinin was used as a loading control. Note the marked increase in hDlg levels in the soluble fraction with concomitant decrease in the insoluble fraction following E6/E7 removal, which is also accompanied by a decrease in SGEF levels in this compartment and a similar loss of SGEF in the insoluble compartment is seen following siRNA ablation of hDlg-1 expression. Panel C. HeLa cells were transfected with control siRNA (Luc), siRNA to E6 (si18E6intronic) or siRNA to E6/E7 and were then analysed for levels of expression of SGEF and p53 in total cell lysates after 72 hrs. α-Actinin was used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285591&req=5

ppat-1002543-g006: High levels of RhoG activity in HPV-18 transformed cells is hDlg and HPV -dependent.Panel A. HeLa cells were transfected with siRNAs against luciferase (Luc) control, E6/E7, hDlg or SGEF as indicated. After 72 hrs cell extracts were then incubated with purified GST.ELMO to determine the levels of active RhoG, which was detected by western blotting (upper panel). The lower panels show input levels of total RhoG, hDlg, SGEF, p53 and α-Actinin. Also shown is the Ponceau stain of the membrane showing constant levels of GST.ELMO. The graph shows the quantifications from multiple GST.ELMO pull-downs, and shows the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent +/− SD of four independent experiments. Note the modest decrease in active RhoG following the removal of E6/E7 and the dramatic decrease following removal of hDlg. Panel B. HeLa cells were transfected with control siRNA (Luc) siRNA to E6/E7 (i) or siRNA to hDlg-1 (ii) and were then analysed for the levels of hDlg, SGEF, and p53 expression after 72 hrs in the insoluble and soluble compartments of the cell. α-Actinin was used as a loading control. Note the marked increase in hDlg levels in the soluble fraction with concomitant decrease in the insoluble fraction following E6/E7 removal, which is also accompanied by a decrease in SGEF levels in this compartment and a similar loss of SGEF in the insoluble compartment is seen following siRNA ablation of hDlg-1 expression. Panel C. HeLa cells were transfected with control siRNA (Luc), siRNA to E6 (si18E6intronic) or siRNA to E6/E7 and were then analysed for levels of expression of SGEF and p53 in total cell lysates after 72 hrs. α-Actinin was used as a loading control.
Mentions: Having shown that hDlg influences the activity of SGEF and RhoG, we wanted to determine what effects HPV-18 E6 might have upon this. To do this we first analysed the levels of RhoG activity in HPV-18 positive cervical tumour derived HeLa cells. GST-ELMO pull-downs were done using extracts from cells in which E6/E7 and hDlg1 expression had been ablated by siRNA transfection. The results and quantitations from multiple assays in Figure 6A show a number of interesting features. Firstly, RhoG activity levels are constitutively high in the control transfected cells. Ablation of E6/E7 expression results, as expected, in an increase in the total levels of hDlg expression and also, most surprisingly, results in a concomitant decrease in the levels of active RhoG. However, ablation of the residual hDlg alone, by siRNA transfection or in combination with siRNA E6/E7 also results in a dramatic decrease in the levels of active RhoG. These results suggest that high levels of RhoG activity in HeLa cells are dependent upon the presence of both E6/E7 and hDlg, even though certain pools of hDlg are also degraded by the HPV-18 E6 oncoprotein. This would therefore suggest that E6 is potentially recruiting or preserving a pool of hDlg that contributes to maintaining elevated SGEF activity. To investigate this possibility we analysed the levels of hDlg expression in the NP-40 soluble and insoluble pools of HeLa cells following ablation of E6/E7 expression. The results in Figure 6B(i) show that, as expected, there is a significant increase in the levels of hDlg expression in the NP-40 soluble fraction upon removal of E6//E7 expression. However, in the insoluble fraction there is a significant decrease in the levels of both hDlg and SGEF expression, suggesting that whilst certain pools of hDlg are targeted for degradation by E6, others are either actively maintained or unaffected. We also wanted to determine if the pattern of SGEF expression in other HPV positive cells was similarly dependent upon E6/E7 expression. To do this we analysed HPV-16 positive CaSki cells. The results in Figure S2 confirm that loss of E6/E7 expression also results in a loss of SGEF from the NP-40 insoluble pool in these cells.

Bottom Line: We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity.In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6.Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

ABSTRACT
A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

Show MeSH
Related in: MedlinePlus