Figure 3.
NPM1 mutants are "born to be exported." (A) A new NPM1 mutation at exon 5. IHC highlights leukemic blasts with cytoplasmic NPM1 (arrows) and normal erythroid cells with a nucleus-restricted expression of NPM1 (asterisk) (bone marrow biopsy, immune-alkaline phosphatase staining using anti–N terminus antibody, clone 376; original magnification ×400) (upper left panel). Sequence of exon 12 of NPM1 was wild-type (data not shown) (lower left panel). Targeted sequencing of the other NPM1 gene exons revealed a new mutation at exon 5, consisting of an in-frame 27-nucleotide insertion and leading to a mutant protein that is 9 aa (shown in red) longer than the wild-type (NPM1wt). Analysis of the new protein sequence predicted a newly acquired NES. Schematic representations of the new mutant protein (NPM1mut-Ex5) compared with wild-type (NPM1wt) (upper right panel). Both are recognized by the anti–N terminus monoclonal antibody (clone 376). The C terminus of the new mutant is unchanged. Cloning of the new mutant NPM1 gene sequence into a GFP-containing vector and expression in NIH3T3 cells confirmed the cytoplasmic localization of the NPM1 mutant protein (GFP-NPM1mut-Ex5), similarly to NPM1 mutant A (GFP-NPM1mutA) (lower right panel). Nucleolar localization of NPM1wt is shown as control (GFP-NPM1wt) (fluorescence microscopy, original magnification ×400). (B) Tuning of NES motif strength. When the capability of NPM1 mutant to bind the nucleolus is completely abrogated (loss of W288 and W290), a weaker C-terminal NES is inserted. When the capability of NPM1 mutant to bind the nucleolus is partially retained (loss of W290 alone), a stronger C-terminal NES motif is inserted. The 3-dimensional reconstruction in the upper left panel is from Falini et al.7