Altered phosphorylation at specific sites confers a mutant phenotype to SV40 wild-type large T antigen in a flat revertant of SV40-transformed cells.

The Rev2 cell line is a cellular revertant of the SV40 wild-type transformed rat cell line SV-52 [Bauer, M., Guhl, E., Graessmann, M. & Graessmann, A. (1987). J. Virol., 61, 1821-1827]. To characterize the level of cellular interference with the SV40 large T antigen (large T)-induced transformation pathway in Rev2 cells, we analysed the biological and biochemical properties of large T expressed in Rev2 cells. We found that Rev2 cells encoded an authentic wild-type large T, with regard to its sequence and its transforming functions. No differences were found in the metabolic stability of large T, or in complex formation with the cellular p53 protein, or in p53 metabolic stabilization. In contrast to SV-52 cells, Rev2 cells showed no association of large T with the chromatin fraction of isolated nuclei. This difference correlated with a reduced affinity of the Rev2 large T to SV40 DNA in vitro. The T proteins from both cell lines were phosphorylated at the same multiple sites. However, in Rev2 cells the phosphorylation of large T at specific serine -residues was significantly reduced. Thus the revertant phenotype of Rev2 cells may be due to an altered phosphorylation state of its large T protein, leading to altered nuclear localization and reduced transforming activity. The alterations of Rev2 large T properties and phosphorylation were very similar to the changes observed with mutant large T in FR(tsA58)A cells, an SV40 tsA58 N-type transformant, when the cells had reverted to the normal phenotype at the non-permissive growth temperature. Thus altered phosphorylation might provide a common structural basis for the biological inactivation of the large T proteins in these cells.