SV40 large T antigen NLS

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126-132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reduced in vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120 derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.