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Comparative Study
. 1996 Nov 12;93(23):12879-84.
doi: 10.1073/pnas.93.23.12879.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells

J Ghai  1 , R S Ostrow, J Tolar, R C McGlennen, T D Lemke, D Tobolt, Z Liu, A J Faras
Affiliations
Comparative Study

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells

J Ghai et al. Proc Natl Acad Sci U S A. .

Abstract

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3'-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

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Figures

Figure 1
Figure 1
Determination of p21ras-bound guanine nucleotides from immunoprecipitates of normal and transformed NIH 3T3 cells. (A) Labeled cells were disrupted following growth under steady-state (SS), EGF-stimulated (+), or quiescent (−) conditions. Anti-p21ras antibody was used to immunoprecipitate p21ras and bound nucleotide. Following dissociation, nucleotides were separated using polyethyleneimine-cellulose chromatography and autoradiographed. The positions of the origin, GDP, and GTP are shown. (B) Anti-p21ras antibody Western blot of anti-p21ras immunoprecipitated cell lysates. Arrow indicates position of p21ras. (C) Independent additional clones of NIH 3T3 (clone 1b), RhPV 1 E7 (clones 3 and 5), and RhPV 1 E5 (clones 1 and 4) cells were tested in duplicate assays for Ras activation under various conditions as indicated with standard error bars shown.
Figure 2
Figure 2
Activation of the PIK in papillomavirus E5-transformed cell lines. Confluent cells were maintained in serum-free media for 24 hr and then harvested for protein, with or without stimulation by EGF. (A) The total cellular protein was immunoprecipitated with an anti-EGFR antibody or a nonspecific antibody (rabbit anti-rat IgG), and protein A, and the PIK assay performed. Results are shown for two independent clones RhPV 1 E5(2) and RhPV 1 E5(4). (B) Distinction between PI-3′-K (type I) and the closely related PI-4′-K (type II) was determined by the former enzyme’s sensitivity to 0.6% Nonidet P-40. The position of polyphosphoinositides and the loading strip are indicated. Intermediate bands represent polyphosphatidylinositols (49).
Figure 3
Figure 3
Sample Western blot analyses for PDGFR and phosphotyrosine-PDGFR. Starved cells were either mock-stimulated (lanes 1, 4, and 7), stimulated with hrPDGF-BB (lanes 2, 5, and 8), or EGF (lanes 3, 6, and 9). Cells were lysed and equal amounts of protein loaded onto a SDS/7.5% polyacrylamide gel. After Western transfer, the blots were tested successively for PDGFR-B (A) and phosphotyrosine (B) analysis. Lanes: 1–3, NIH 3T3; 4–6, RhPV 1 E7; 7–9, RhPV 1 E5. Molecular weight markers in kDa are shown on the left and the receptor locations indicated.
Figure 4
Figure 4
Relative EGFR, PDGFR, and phosphotyrosine to PDGFR ratios. Cells were treated as in Fig. 3 to detect EGFR (A), PDGFR (B), or phosphotyrosine and PDGFR (C) ratios. Using PhosphorImager analysis, the intensity of each band (less background) in A and B was normalized to the intensity of the unstimulated NIH 3T3 (NIH 3T3 + BSA) cells for that blot. In C, the intensity of phosphotyrosine signal comigrating with PDGFR was compared with the intensity of the PDGFR signal from the same blot and normalized to the ratio obtained from unstimulated NIH 3T3 cells. Error bars show the standard error of the mean of the ratios. Results are from three independent experiments, and blots of two were in duplicate.
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