The Ad5 E1A 12S gene encodes an oncoprotein with the ability to immortalize and cooperate with other viral or cellular oncoproteins to transform primary epithelial cells. The immortalizing function is dependent on the protein's efficient localization to the nucleus. A five amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro, has been identified at the extreme COOH-terminus. This signal is necessary but not sufficient for efficient nuclear localization. A mutational analysis has been undertaken to further characterize the 12S NLS. The individual amino acids of the signal appear to have varying functional relevance. The lysine residue (a.a. 239) and the first arginine residue (a.a. 240) are the most critical. Changing the second arginine (a.a. 242) to threonine or either proline (a.a. 241 or 243) to alanine marginally diminishes signal function. Replacing the 12S NLS with the SV40 large T antigen (LT) NLS does not measurably affect the protein's nuclear localization. Sequences directly upstream of the NLS have a significant role in the proper localization of the 12S protein as illustrated by inefficiently localized mutants that have deletions of these sequences. Analyses of these mutants using a monoclonal antibody that recognizes the COOH-terminal four amino acids of the NLS have revealed that their signals are probably masked. To further investigate the importance of protein context in signal function, several NLS insertion mutants were constructed. Two regions in the first exon with predicted high surface probabilities and no known functions were chosen as sites for NLS insertions. Neither a wild-type 12S- nor a SV40 LT-NLS was functional in any of the new locations, indicating that for 12S, positioning of the NLS in the protein is critical.
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