Efficient nuclear localization of the Ad5 E1A 12S protein is necessary for immortalization but not cotransformation of primary epithelial cells.

The Ad5 E1A 12S gene encodes a nuclear protein that can immortalize and cooperate with other oncoproteins to transform primary epithelial cells. Expression of the first exon is necessary for the activation of cellular proliferation, immortalization, and transformation. The second exon is necessary for the maintenance of cellular proliferation, immortalization, and the induction of an epithelial cell growth factor but is dispensable for cotransformation with T24 ras. Expression of the second exon is also necessary for efficient nuclear localization of the 12S polypeptide. A five-amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro is encoded by the last 15 nucleotides of the second exon. To determine the role of subcellular localization in immortalization and transformation, a mutational analysis of the second exon and the 12S NLS was undertaken. The results from our analysis indicate that regions of the second exon outside the NLS affect nuclear localization. These regions necessary for efficient nuclear localization of the 12S protein are coincident with regions necessary for immortalization. In contrast, these regions are dispensable for cotransformation with T24 ras. The importance of the positively charged amino acids of the NLS in signal function and immortalization is varied. Lys 239 is the most critical residue, followed by Arg 240 and Arg 242, respectively. These data indicate that efficient nuclear localization of 12S is necessary for immortalization but not cotransformation with T24 ras.