Figure 6. HPV16 E7 expression activates NHE1 via a PKA-mediated-reduction in RhoA activity.

A. Representative Western Blot of three GST-RBD pull-downs showing RhoA activity in 2BN11 cells at different times after tet removal and treated with 100 nM H89 and 10 µM FSK for the indicated times. The lower gel shows the amount of total RhoA in cell lysates. B. Sensitized FRET measurements of 2BN11 cells to determine the effect of E7 expression on RhoA activity in live cells. Cells were transfected with the RhoA biosensor pRaichu-1297× and then cultured with or without tet for 24 hr and treated or not with 100 nM H89 during the 24 hr period. Cells were imaged for CFP and FRET and the relative decreases in CFP/FRET ratios obtained (presented in the central column) indicate a decrease in active RhoA. Data are the mean±SEM from 32 to 37 different cells. ***P<0.005. CFP/FRET ratio images are in pseudocolor, with the color indicating the relative value at each pixel (lateral images), such that blue indicates the highest EmCFP/EmYFP ratio (highest RhoA activity), and red reflects the lowest EmCFP/EmYFP ratio (lowest RhoA activity). Scale bar is 10 µm. C. Role of PKA-mediated RhoA signalling on HPV16 E7-induced up-regulation of NHE1 activity. 2BN11 cells were transfected transiently with cDNA for either a phosphorylation dead (pd) RhoA mutant (blue cross-hatched bars), with dominant negative RhoA (dn) mutant (green stippled bars), with constitutively active RhoA (ca) (red striped bars) mutant or with siRNA against RhoA (brown reverse stippled bars). Control cells were transfected with the empty plasmid or non-specific siRNA transfected cells (scrambled) served as the control for RhoA silencing. After 24 hrs for cDNA construct or 48 hrs for siRNA transfection, tetracycline was then removed (−tet) or not (+tet) for a further 24 hrs and NHE1 activity was measured as described in Materials and Methods. Expression of these constructs had no effect on basal NHE1 activity in control, +tet cells. Inactivation of RhoA with the dn mutant and siRNA significantly potentiated the E7-induced stimulation of NHE1 activity while both activation of RhoA with the ca mutant and the block of RhoA phosphorylation by PKA with the pd mutant abrogated the E7-induced stimulation of NHE1 activity. Efficiency of siRNA-mediated RhoA knock-down was analyzed with immunofluorescence assay by using a monoclonal anti-RhoA antibody (green) and the blue fluorescent dye DAPI for staining nuclei (Figure S2).