Nuclear and cytoplasmic localisation of the leukemia associated nup98-rap1gds1 fusion protein

We have identified a new leukemia fusion gene, NRG, which is created by fusion of the 5′ end of the NUP98 gene to the entire coding sequence of the RAP1GDS1 gene. The translocation giving rise to NRG, t(4;ll)(q21;pl5), is recurrent in adult T-cell acute lymphocytic leukemia. In order to begin to understand the mechanism by which NRG causes leukemia, we set out to determine the cellular location of the hybrid protein nrg and its components nup98t (t for truncated) and smgGDS (the protein encoded by RAP1GDSI). cDNAs encoding nup98, nup98t, smgGDS and nrg were cloned into the pEGFP-C2 mammalian expression vector to create proteins tagged at their amino-terminus with green fluorescent protein (gfp). The location of the gfp tagged proteins within transfected NIH3T3 and HEK-293 cells was visualised using confocal microscopy. Nup98 and nup98t were located in a punctate pattern around the nuclear envelope. SmgGDS was found throughout the cytoplasm and was absent from the nucleus. The hybrid protein nrg was present throughout the cytoplasm and was also visible within specific subnuclear domains in a pattern similar to a number of proteins with known or putative roles in transcription. In some cells nrg showed a marked bias toward either a nuclear or a cytoplasmic localisation. This suggests that nrg may shuttle between the nucleus and the cytoplasm, perhaps in a cell cycle dependent fashion. The 5' portion of nrg, nup98t, has been shown to have strong transcriptional transactivation ability through its interaction with CBP/p300. The smgGDS portion of nrg is comprised of 12 tandem repeats of the armadillo motif, which is generally thought to act as a protein-protein interaction motif. Therefore the (4;ll)(q21;pl5) translocation results in aberrant nuclear localisation of the smgGDS moiety of nrg and effectively fuses a transcriptional transactivation domain to a proteinprotein interaction domain. In the nucleus nrg may act as a bi-partite transcriptional regulator by recruiting the transcriptional coactivators CBP/p300 and an unidentified DNA binding protein. In the cytoplasm, smgGDS has been shown to be important in blocking apoptosis in thymocytes. It is therefore possible that nrg promotes leukemogenesis through two independent pathways. Nrg may act in the nucleus to deregulate transcription pathways critical to normal T-cell development, while in the cytoplasm, nrg may act to increase T-cell survival by promoting an anti-apoptotic phenotype.