Nuclear import and subnuclear localization of the proto-oncoprotein ETO (MTG8).
ETO (MTG8) was first described due to its involvement in the (8;21) translocation frequently observed in acute myeloid leukemias. In the t(8;21) the AML1 gene on chromosome 21 is fused to ETO on chromosome 8. The resultant hybrid protein is comprised of the DNA binding domain of AML-1 and the majority 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 portion of ETO (amino acids 241-280) that was sufficient to cause nuclear import of green fluorescent protein. Mutational analysis demonstrated that lysine 265 and/or arginine 266 were required for nuclear import 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 interaction with importin-alpha and beta. These data suggest that ETO enters the nucleus via an importin-mediated pathway. Additionally, ETO and AML-1/ETO co-localized to punctate nuclear bodies distinct from those containing promyelocytic 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.
Odaka
Y
Y
Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center School of Medicine, Shreveport 71130, USA.
Mally
A
A
Elliott
L T
LT
Meyers
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells
C107844:AML1-ETO fusion protein, human; D000596:Amino Acids; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D000075142:RUNX1 Translocation Partner 1 Protein; C493728:RUNX1 protein, human; C082222:RUNX1T1 protein, human; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D001120:Arginine; D008239:Lysine