Nuclear import and subnuclear localization of the proto-oncoprotein ETO (MTG8)

ETO (MTG8) was first described due to its involvement in the (8;21) translo cation frequently observed in acute myeloid leukemias. In the t(8;21) the A ML1 gene on chromosome 21 is fused to ETO on chromosome 8. The resultant hy brid protein is comprised of the DNA, binding domain of AML-1 and the major ity of ETO. This study examines the subnuclear distributions of ETO, AML-1B and AML-1/ETO proteins fused to green fluorescence protein in living cells using fluorescence microscopy. Further, we identified a 40 amino acid port ion of ETO (amino acids 241-280) that was sufficient to cause nuclear impor t of green fluorescent protein. Mutational analysis demonstrated that lysin e 265 and/or arginine 266 were required for nuclear impart of ETO, but that the surrounding basic residues were not critical, ETO interacted with the nuclear import proteins importin-alpha and beta in vitro, and mutations in ETO that abolish nuclear localization also abolished the in vitro interacti on with importin-alpha and beta. These data suggest that ETO enters the nuc leus via an importin-mediated pathway. additionally, ETO and AMIL-1/ETO co- localized to punctate nuclear bodies distinct from those containing promyel ocytic leukemia protein. Nuclear body formation was dependent upon a region of ETO N-terminal to the nuclear localization signal. Thus, ETO and AML-1/ ETO reside in potentially novel subnuclear compartments.