Construction and characterization of epitope-tagged HPV-16 E7 proteins. (A) Schematic representation of the FLAG/HA double tagged HPV-16 E7 fusion proteins constructed for this study. C-E7 and N-E7 contain FLAG/HA tags fused to their C and N termini, respectively. The amino acid sequences corresponding to the epitope tags are underlined. (B) Transforming activities of C-E7 and N-E7 compared to untagged HPV-16 E7 (E7) as determined by induction of anchorage independent growth in NIH 3T3 cells. Values shown were obtained after subtracting the number of colonies observed with vector transduced NIH 3T3 cells (273), even though these colonies were smaller than those in HPV oncoprotein expressing cells. Averages and standard deviations from an experiments performed with duplicate plates are shown.

Identification of p600 as an interactor of HPV-16 E7 and mapping of E7 domains necessary for p600 association. (A) SDS/4–12% PAGE analysis of cellular protein complexes associated with C-E7 in HeLa cells after TAP. C denotes TAP of an equivalent amount of cell lysates from cells transduced with empty vector. Gel was stained with colloidal blue; protein bands containing C-E7, known HPV-16 E7 associated proteins and p600 are indicated. See text for details. (B) Mapping of the sequences on HPV-16 E7 that are necessary for p600 association. HeLa cells with stable expression of C-E7 and the corresponding E7 mutants double tagged with FLAG/HA at their C termini were subjected to FLAG immunoprecipitation followed by immunoblot analysis for p600 (Top) and pRB (Middle) as a control. Expression of the corresponding E7 mutants is documented by immunoblot using HA antibody (Bottom). The different panels are derived from the same gel and x-ray film. (C) The low-risk HPV-6b and HPV-11 E7 proteins associate with p600 as efficiently as HPV-16 E7. HeLa cells with stable expression of the corresponding E7 proteins tagged with FLAG/HA at their C termini were subjected to FLAG immunoprecipitation followed by immunoblot analysis for p600 (Top) and pRB (Middle). Expression of the corresponding E7 mutants is documented by immunoblot using HA antibody (Bottom).

Association and colocalization of HPV-16 E7 with p600 in cervical cancer cells. (A) HPV-16 E7 associates with p600 in the HPV-16 positive CaSki human cervical carcinoma cell line. Immunoprecipitation was performed with E7-specific antibodies followed by detection of p600 by immunoblot. The HPV-negative cervical cancer cell line C33 expresses similar p600 levels as CaSki and was used as a specificity control for these experiments. (B) Localization of HPV-16 E7 (green, Left) and p600 (red, Center) in CaSki cells in nuclear and cytoplasmic structures by confocal fluorescence microscopy. The merged picture (yellow, Right) documents colocalization of the two proteins. (C) Depletion of p600 (Top) by stable expression of a p600-specific shRNA expression plasmid (600i) in HPV-16 E7 expressing IMR-90 human diploid fibroblasts (Left) and the HPV-16-positive CaSki cervical cancer cell line (Right) does not affect intracellular pRB levels (Middle). GAPDH protein levels (Bottom) are shown to document equal loading. Cells expressing the reverse orientation shRNA expression vector were used as controls (Ci).

Depletion of p600 by stable expression of specific shRNA expression plasmids (600i) decreases anchorage independent growth in HPV-16 positive CaSki cervical cancer (A), HPV-16 E7 expressing NIH 3T3 (B), and HPV-16 E6/E7 expressing NIH 3T3 (C) and HPV-negative U2OS osteosarcoma (D) cells. Decreased p600 expression is documented by immunoblot; GAPDH protein levels are shown as a loading control. Cells expressing the reverse orientation shRNA expression vector were used as controls (Ci). The bar graphs denote averages and standard deviations of three (NIH 3T3 E7, E6/E7) or two (CaSki, U2OS) experiments, each performed with duplicate plates.