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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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Related in: MedlinePlus

HPV18E6 does not degrade the NP-40 insoluble pool of hDlg in the presence of SGEF.HEK293 cells were transfected with HA-tagged Dlg, Myc-tagged SGEF, untagged HPV18E6 expression plasmids either alone or in different combinations as indicated. After 24 hrs the cells were extracted into NP-40 soluble and NP-40 insoluble fractions and the patterns of expressions of Dlg, SGEF and E6 determined by western blot analysis. β-gal expression was used as a marker for transfection efficiency and a loading control.
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ppat-1002543-g008: HPV18E6 does not degrade the NP-40 insoluble pool of hDlg in the presence of SGEF.HEK293 cells were transfected with HA-tagged Dlg, Myc-tagged SGEF, untagged HPV18E6 expression plasmids either alone or in different combinations as indicated. After 24 hrs the cells were extracted into NP-40 soluble and NP-40 insoluble fractions and the patterns of expressions of Dlg, SGEF and E6 determined by western blot analysis. β-gal expression was used as a marker for transfection efficiency and a loading control.

Mentions: To investigate this further we performed a series of transient overexpression experiments in 293 cells. The cells were transfected with an untagged E6 expression vector, together with Dlg and SGEF. Cell extracts were divided into NP-40 soluble and insoluble pools and the levels of expression of E6, Dlg and SGEF were analysed by western blotting. The results in Figure 8 demonstrate a number of interesting features. Co-expression of Dlg and SGEF results in a marked accumulation of both proteins in the NP-40 insoluble compartment. When Dlg and E6 are co-transfected, E6 induces a dramatic decrease in the levels of Dlg, particularly in the NP-40 soluble fraction of the cells. However, the levels of Dlg are largely unaffected by E6 in the NP-40 insoluble pool when SGEF is also present. Furthermore, E6 also becomes localized to this NP-40 insoluble fraction when both SGEF and Dlg are present. Taken together, these results demonstrate that E6, Dlg and SGEF can exist in complex within the NP-40 insoluble fraction of the cell, and that this is dependent upon the ability of E6 to bind Dlg in a PDZ-domain dependent manner.


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)

HPV18E6 does not degrade the NP-40 insoluble pool of hDlg in the presence of SGEF.HEK293 cells were transfected with HA-tagged Dlg, Myc-tagged SGEF, untagged HPV18E6 expression plasmids either alone or in different combinations as indicated. After 24 hrs the cells were extracted into NP-40 soluble and NP-40 insoluble fractions and the patterns of expressions of Dlg, SGEF and E6 determined by western blot analysis. β-gal expression was used as a marker for transfection efficiency and a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002543-g008: HPV18E6 does not degrade the NP-40 insoluble pool of hDlg in the presence of SGEF.HEK293 cells were transfected with HA-tagged Dlg, Myc-tagged SGEF, untagged HPV18E6 expression plasmids either alone or in different combinations as indicated. After 24 hrs the cells were extracted into NP-40 soluble and NP-40 insoluble fractions and the patterns of expressions of Dlg, SGEF and E6 determined by western blot analysis. β-gal expression was used as a marker for transfection efficiency and a loading control.
Mentions: To investigate this further we performed a series of transient overexpression experiments in 293 cells. The cells were transfected with an untagged E6 expression vector, together with Dlg and SGEF. Cell extracts were divided into NP-40 soluble and insoluble pools and the levels of expression of E6, Dlg and SGEF were analysed by western blotting. The results in Figure 8 demonstrate a number of interesting features. Co-expression of Dlg and SGEF results in a marked accumulation of both proteins in the NP-40 insoluble compartment. When Dlg and E6 are co-transfected, E6 induces a dramatic decrease in the levels of Dlg, particularly in the NP-40 soluble fraction of the cells. However, the levels of Dlg are largely unaffected by E6 in the NP-40 insoluble pool when SGEF is also present. Furthermore, E6 also becomes localized to this NP-40 insoluble fraction when both SGEF and Dlg are present. Taken together, these results demonstrate that E6, Dlg and SGEF can exist in complex within the NP-40 insoluble fraction of the cell, and that this is dependent upon the ability of E6 to bind Dlg in a PDZ-domain dependent manner.

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