DOSE-DEPENDENT COMPETITION BETWEEN NPM LEUKEMIC MUTANTS AND ARF PROTEIN FOR SUBCELLULAR DISTRIBUTION: A NUCLEAR TUG-OF-WAR

Acute myeloid leukemia (AML) is frequently targeted by mutations at exon-12 of the nucleophosmin (NPM) gene (Falini et al., NEJM, 352:254, 2005), which 1) disrupt either tryptophan 290 or trytophans 288 and 290, constituting the nucleolar localization signal (NoLS); and 2) cre- ate a new carboxy-terminal Nuclear Export Signal (NES) motif, with 6 variations observed to date. Both abnormalities underlie aberrant NPM accumulation in leukemic cell cytoplasm (NPMc+ AML). In NPM mutants, the new NES motif non-randomly correlates with NoLS dis- ruption at the C-terminus. The most common NES motif (LxxxVxxVxL) always associates with mutations of tryptophans(W) 288 and 290, e.g. mutant A; a NES variant sequence, such as LxxxLxxVxL, always associ- ates with W288 retention in rare NPM mutants, e.g. mutant E (Falini et al., Blood, pub-ahead, February 2, 2006). These findings suggest diverse sequences of mutant NES motifs function differently. Mutated NPM dislocates Arf from nucleoli, shortens its half-life and blunts its func- tions (Den Besten W et al., Cell Cycle, 4:1593, 2006). In different NPM mutants this study addressed: i) the role of variations in NES motifs; and ii) interactions with Arf. i) Role of different NES motifs in altered NPM nucleo-cytoplasmic traffic: Arf-negative NIH-3T3 cells were transfected with eGFP-tagged NPM mutant A in which W288 had been artificially inserted by site-directed mutagenesis (eGFP-NPMmA_C288W). Unlike mutant E, which is unaffected by the presence of W288, this protein dis- played greatly reduced cytoplasmic export. Additionally, in NPM mutant E, replacing the LxxxLxxVxL NES sequence with LxxxVxxVxL (eGFP- NPMmE_LVVL) partially relocated mutant E to the nucleus. These results demonstrate efficiency differences between NES: LxxxVxxVxL is weaker than LxxxLxxVxL. The former is strong enough to export the NPM mutants only if both tryptophans are mutated whilst LxxxLxxVxL is needed if W288 is retained. ii) Interaction of NPM mutants with Arf: We investigated how changes at the NPM mutant C-terminus influence NPM-Arf binding, and NPM and Arf subcellular distribution. Arf-nega- tive NIH-3T3 cells were co-transfected with DsRed-tagged Arf (DsRed- monomer-Arf) and eGFP-tagged NPM mutants. Arf partially relocated NPM mutants A and E from cytoplasm to nucleus in a dose-related man- ner. In turn, NPM mutants partially relocated Arf from the nucleolus to nucleoplasm and cytoplasm. These results demonstrate a reciprocal interaction between Arf and NPM mutants. Moreover lower doses of Arf completely relocated artificial mutants eGFP-NPMmA_C288W and eGFP-NPMmE_LVVL to the nucleus, suggesting these artificial mutants have a stronger affinity for Arf than NPM mutants A and E. Co-immuno- precipitation studies showed mutants A and E bind less Arf than wild- type NPM or eGFP-NPMmA_C288W and eGFP-NPMmE_LVVL. Conclu- sions. The non-random correlation between NPM NoLS disruption and NES sequence variants is feasibly explained by need for 1) efficient cyto- plasmic accumulation of mutated NPM, and 2) less efficient binding of mutant NPM to Arf as compared to wild-type NPM. Both mechanisms may contribute to Arf dislocation/degradation, thus having the same functional consequences as NPM silencing. These findings may be rel- evant to the pathogenesis of NPMc+ AML.