SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence

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 (Ser(120)) 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 Ser(120) 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 Asp(120) and Ala(120) 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 Asp(120) protein compared with the Ala(120) protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import, The Asp(120) protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser(120) may also regulate the nuclear import rate, CKII phosphorylation of the Asp(120) protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser(120) and Ala(120) proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser(120) 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 Ala(120) protein derivative had a binding affinity very similar to that of wild type, the Asp(120) 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.