A BIPARTITE NUCLEAR-LOCALIZATION SIGNAL IN THE RETINOBLASTOMA GENE-PRODUCT AND ITS IMPORTANCE FOR BIOLOGICAL-ACTIVITY

The retinoblastoma gene product, p110RB1, appears to regulate cell gro wth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic reg ion, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequen ce similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give m utant NLS(NQ)] or deletion of the entire NLS (DELTANLS) abrogated excl usive nuclear localization, yielding proteins which were distributed e ither equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/DELTA22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Esc herichia coli 13-galactosidase, the RB1 NLS directed this protein to t he nucleus, indicating that the motif is not only necessary but also s ufficient for nuclear transport. Neither NLS(NQ) nor DELTANLS was hype rphosphorylated in vivo, but both retained their abilities to interact , in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple co py number, the NLS mutant alleles displayed reduced biological activit y, measured by inhibition of growth of the osteogenic sarcoma cell lin e Saos-2, which has no wild-type RB1. Naturally occurring mutations an d deletions in exon 25 of RB1 which disrupt the NLS may lead to partia l or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.