Regions and activities of simian virus 40 T antigen that cooperate with an activated ras oncogene in transforming primary rat embryo fibroblasts

Abstract

Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

FIG. 1.

Cooperation between T-antigen fragments and Ras. Each bar represents the average number of dense foci counted in duplicate flasks transfected with a ras oncogene expression plasmid and the DNA indicated. Different types of shading of the bars represent independent experiments. The results are expressed as a percentage of the number of foci produced by T1-708 and Ras. The average number of foci in flasks transfected with the T1-708 expression plasmid ranged from 81 to 142 in the individual experiments.

FIG. 2.

Effect of J-domain and Rb-binding-region mutations on Ras cooperation. Each bar represents the average number of dense foci counted in duplicate flasks transfected with a ras oncogene expression plasmid and the DNA indicated. Different types of shading of the bars represent independent experiments. The results are expressed as a percentage of the number of foci produced by the T1-708,t expression vector and Ras. The average number of foci in flasks transfected with the T1-708,t expression vector ranged from 50 to 103 in the individual experiments.

FIG. 3.

Transactivation of the cyclin A promoter is a J-domain-dependent, Rb-binding-independent T-antigen activity. TC7 cells were transfected with the reporter pAluc and the DNA indicated. (A) Transient transfection assays performed with medium containing high or low concentrations of serum (hs or ls, respectively). (B) Results obtained with hs. Since transactivation levels are influenced by the plasmid backbone of constructs expressing T antigen (7), the mutants in each experiment were examined in relation to wild-type T antigen expressed from the same plasmid backbone.

FIG. 4.

Ability of T antigens to overcome Rb-induced growth arrest. In each of duplicate dishes, the number of large cells in a total of 1,000 cells was determined. The insert shows the morphology of cells transfected with Rb and T1-127 (left) and Rb and T1-127E107K (right) expression plasmids.

FIG. 5.

Effect of mutations in the p53-binding region on Ras cooperation. (Top) Positions of p53-binding regions and amino acid substitutions examined. For each mutant tested, the amino acid substitution and position within T antigen are indicated. (Bottom) Ability of a mutant to cooperate with Ras, indicated by a plus or a minus sign and quantitatively as a percentage of the wild-type value; values are expressed as the average of three or more experiments. The percentage of the wild-type value for the T251-708 segment (49%) is the average value obtained from six experiments. The average number of foci in flasks transfected with the T1-708 expression plasmid ranged from 80 to 168 in the individual experiments.

FIG. 6.

T251-708 proteins with amino acid substitutions in the p53-binding region that do not cooperate with Ras retain the ability to inhibit p53 as a transcriptional transactivator. (A) Effect of adding Bcl2 to transient transfection-luciferase assays involving p53. Error bars indicate standard deviations. (B) Luciferase assays of cells transfected with the mutant indicated, Bcl2, wild-type p53, and the reporter construct. Narrow bars indicate standard deviations. (C) Immunoprecipitation and Western blot analysis of the T-antigen levels in parallel cultures of transfected cells. Equal amounts of protein were immunoprecipitated with PAb901, which recognizes an epitope in the C terminus of T antigen in all cases except T1-127. T1-127 protein was immunoprecipitated with PAb416, which recognizes an epitope in the N terminus of T antigen adjacent to but not including the region common to T antigen and t antigen; this antibody does not recognize small t antigen.

FIG. 7.

Diagram indicating T-antigen activities that are dependent on the J domain, the Rb-binding motif, or both. Data were obtained from the following sources: transactivation of cyclin A (this study); activation of Hsc70 ATPase (58); override of p53 growth arrest (16, 46); anchorage-independent cell division, growth in medium containing low serum concentrations, and growth to high cell density (61, 65); binding and enhancement of Tst-1/Oct/SCIP (55); disruption of p103-E2F complexes, relief of Rb/p130-dependent E2F repression, and override of Rb/p107/p130-mediated growth arrest (70); hypophosphorylation of p107/p130 (61); degradation of p130 (60); and cooperative transformation with Ras by an N-terminal fragment of T antigen (6). Asterisks indicate divergence in results from different studies.