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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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Dlg enhances RhoG activity in an SGEF dependent manner.Panel A. HEK293 cells were either transfected with vector or HA-tagged Dlg and Flag-tagged SGEF as indicated. After 24 hrs cell extracts were made which were then incubated with purified GST-ELMO to pull down active RhoG which was detected by western blot analysis. The three lower panels show total protein inputs for RhoG, Dlg and SGEF. Panel B. Graph showing the quantification from multiple GST-ELMO pull-downs, showing the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent ±SD of three independent experiments Panel C. Extracts from HaCaT cells stably ablated for hDlg expression (shDlg) either with or without Dlg rescue expression were used in a GST-ELMO pulldown assay to determine RhoG activity. Extracts from untreated HaCaT cells or HaCaT cells stably expressing control shRNA(TR2) were used as control.
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ppat-1002543-g005: Dlg enhances RhoG activity in an SGEF dependent manner.Panel A. HEK293 cells were either transfected with vector or HA-tagged Dlg and Flag-tagged SGEF as indicated. After 24 hrs cell extracts were made which were then incubated with purified GST-ELMO to pull down active RhoG which was detected by western blot analysis. The three lower panels show total protein inputs for RhoG, Dlg and SGEF. Panel B. Graph showing the quantification from multiple GST-ELMO pull-downs, showing the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent ±SD of three independent experiments Panel C. Extracts from HaCaT cells stably ablated for hDlg expression (shDlg) either with or without Dlg rescue expression were used in a GST-ELMO pulldown assay to determine RhoG activity. Extracts from untreated HaCaT cells or HaCaT cells stably expressing control shRNA(TR2) were used as control.

Mentions: SGEF has been shown to be a GEF specific for RhoG [45]. To test whether the interaction and the recruitment of SGEF to the cytoskeleton by Dlg has any biological consequences in terms of SGEF regulation of RhoG, we proceeded to monitor the levels of RhoG activity, as determined by its ability to interact with the downstream target, ELMO [46]. To do this we performed an affinity pulldown that specifically precipitates active GTP-bound RhoG, as previously described [45]. The results obtained in Figure 5A, together with the quantifications from multiple assays in Figure 5B, show no alteration in total RhoG levels under the different assay conditions, whilst there is a significant increase in the levels of ELMO-bound active RhoG in cells transfected with SGEF, and this is in agreement with previous studies [45]. However, most strikingly, co-transfection of Dlg and SGEF results in a dramatic increase in the amount of active RhoG, suggesting that Dlg can increase SGEF activity with respect to its downstream effector, RhoG. As an additional verification of these studies, we also monitored RhoG activity in a similar manner in cells in which hDlg expression had been stably ablated. The results of the pull-down assays are shown in Figure 5C and clearly show that, whilst there are no changes in total levels of RhoG expression, there is a greatly reduced level of active RhoG present within the hDlg-depleted cells in comparison with the different sets of control cells, and this increases in the rescued rat Dlg-expressing cells. In conclusion, these data demonstrate that hDlg positively regulates SGEF- induced RhoG activity in epithelial 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)

Dlg enhances RhoG activity in an SGEF dependent manner.Panel A. HEK293 cells were either transfected with vector or HA-tagged Dlg and Flag-tagged SGEF as indicated. After 24 hrs cell extracts were made which were then incubated with purified GST-ELMO to pull down active RhoG which was detected by western blot analysis. The three lower panels show total protein inputs for RhoG, Dlg and SGEF. Panel B. Graph showing the quantification from multiple GST-ELMO pull-downs, showing the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent ±SD of three independent experiments Panel C. Extracts from HaCaT cells stably ablated for hDlg expression (shDlg) either with or without Dlg rescue expression were used in a GST-ELMO pulldown assay to determine RhoG activity. Extracts from untreated HaCaT cells or HaCaT cells stably expressing control shRNA(TR2) were used as control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285591&req=5

ppat-1002543-g005: Dlg enhances RhoG activity in an SGEF dependent manner.Panel A. HEK293 cells were either transfected with vector or HA-tagged Dlg and Flag-tagged SGEF as indicated. After 24 hrs cell extracts were made which were then incubated with purified GST-ELMO to pull down active RhoG which was detected by western blot analysis. The three lower panels show total protein inputs for RhoG, Dlg and SGEF. Panel B. Graph showing the quantification from multiple GST-ELMO pull-downs, showing the fold change in the levels of RhoG activity under the different experimental conditions. Error bars represent ±SD of three independent experiments Panel C. Extracts from HaCaT cells stably ablated for hDlg expression (shDlg) either with or without Dlg rescue expression were used in a GST-ELMO pulldown assay to determine RhoG activity. Extracts from untreated HaCaT cells or HaCaT cells stably expressing control shRNA(TR2) were used as control.
Mentions: SGEF has been shown to be a GEF specific for RhoG [45]. To test whether the interaction and the recruitment of SGEF to the cytoskeleton by Dlg has any biological consequences in terms of SGEF regulation of RhoG, we proceeded to monitor the levels of RhoG activity, as determined by its ability to interact with the downstream target, ELMO [46]. To do this we performed an affinity pulldown that specifically precipitates active GTP-bound RhoG, as previously described [45]. The results obtained in Figure 5A, together with the quantifications from multiple assays in Figure 5B, show no alteration in total RhoG levels under the different assay conditions, whilst there is a significant increase in the levels of ELMO-bound active RhoG in cells transfected with SGEF, and this is in agreement with previous studies [45]. However, most strikingly, co-transfection of Dlg and SGEF results in a dramatic increase in the amount of active RhoG, suggesting that Dlg can increase SGEF activity with respect to its downstream effector, RhoG. As an additional verification of these studies, we also monitored RhoG activity in a similar manner in cells in which hDlg expression had been stably ablated. The results of the pull-down assays are shown in Figure 5C and clearly show that, whilst there are no changes in total levels of RhoG expression, there is a greatly reduced level of active RhoG present within the hDlg-depleted cells in comparison with the different sets of control cells, and this increases in the rescued rat Dlg-expressing cells. In conclusion, these data demonstrate that hDlg positively regulates SGEF- induced RhoG activity in epithelial 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