STRUCTURAL LIMITATIONS OF THE AD5 E1A 12S NUCLEAR-LOCALIZATION SIGNAL

The Ad5 EIA 125 gene encodes an oncoprotein with the ability to immort alize and cooperate with other viral or cellular oncoproteins to trans form 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 analys is has been undertaken to further characterize the 12S NLS. The indivi dual amino acids of the signal appear to have varying functional relev ance. 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 marginall y diminishes signal function. Replacing the 12S NLS with the SV40 larg e T antigen (LT) NLS does not measurably affect the protein's nuclear localization. Sequences directly upstream of the NLS have a significan t role in the proper localization of the 12S protein as illustrated by inefficiently localized mutants that have deletions of these sequence s. Analyses of these mutants using a monoclonal antibody that recogniz es the COOH-terminal four amino acids of the NLS have revealed that th eir 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 su rface probabilities and no known functions were chosen as sites for NL S insertions. Neither a wild-type 12S- nor a SV40 LT-NLS was functiona l in any of the new locations, indicating that for 12S, positioning of the NLS in the protein is critical. (C) 1996 Academic Press, Inc.