Kinetic properties of nuclear transport conferred by the retinoblastoma (Rb) NLS

Abstract

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein central to control of cellular proliferation. We have previously shown that human RB possesses an evolutionary conserved bipartite nuclear localization sequence (NLS) (KRSAEGSNPPKPLKKLR877) resembling that of nucleoplasmin. Here we analyze the kinetic properties of the RB NLS in detail with respect to recognition by cellular nuclear import factors, the importins (IMPs), and nuclear transport properties, comparing results to those for the NLSs from SV40 large tumor antigen (T-ag) and the Xenopus laevis phosphoprotein N1N2. Binding affinities of different IMPα subunits for the Rb NLS, in the absence or presence of IMPβ subunits were determined, and NLS-dependent nuclear import reconstituted in vitro for the first time using purified IMPα/β subunits together with recombinant human RanGDP and nuclear transport factor 2 (NTF2). RB NLS-mediated transport had a strict requirement for all components, with high NTF2 concentrations inhibiting transport. As in the case of transport mediated by the T-ag- and N1N2-NLSs, nuclear import of an RB-NLS containing β-Gal fusion protein was reduced or abolished when anti-IMPα or β antibody was added to cytosolic extract, respectively, confirming that RB NLS-mediated nuclear import occurs through action of IMPα/β. We conclude that although mediated by IMPα/β, and similar in most respects to transport mediated by the similarly bipartite N1N2 NLS, nuclear import conferred by the RB NLS has distinct properties, in part due to the affinity of its interaction with IMPα.