Born to Be Exported: COOH-Terminal Nuclear Export Signals of Different Strength Ensure Cytoplasmic Accumulation of Nucleophosmin Leukemic Mutants

Abstract

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH 2 -terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis. [Cancer Res 2007;67(13):6230-7]

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

“…Interaction of Crm1 (exportin 1) with the two NPM1wt N-terminus NES motifs 13,14 (which presumably occurs both in nucleoplasm and nucleolus) ensures NPM1wt nuclear export. Using a Rev(1.4)-based shuttling assay, we found that both NPM1wt NES motifs have low export efficiency 41 and, therefore, little native protein is exported from nucleus to cytoplasm. As NLS-mediated nuclear import of NPM1wt greatly predominates over export, 41 most NPM1wt resides within the nucleolus (Figure 2).…”

“…Using a Rev(1.4)-based shuttling assay, we found that both NPM1wt NES motifs have low export efficiency 41 and, therefore, little native protein is exported from nucleus to cytoplasm. As NLS-mediated nuclear import of NPM1wt greatly predominates over export, 41 most NPM1wt resides within the nucleolus (Figure 2). In contrast, the rare NPM1.2 (or B23.2) truncated isoform localizes to nucleoplasm, 8 as it lacks the C-terminus nucleolar-binding domain and cannot, therefore, bind efficiently to the nucleolus.…”

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…This new NES is added to and reinforces the two NES normally present in the N-terminal domain of the native protein. 27 Notably, when only Trp290 is replaced, a stronger NES motif is inserted to ensure maximal export in Figure 3. Mutations at the C-terminal domain are associated with acute myeloid leukemia.…”

“…27 Very rarely, mutations may occur at exon XI of NPM1 leading to a mutant protein that is truncated at its C-terminus and lacks both tryptophans. 28 The combined effect of the loss of either one or both tryptophan residues and the appearance of a new NES in the variant protein is mandatory to completely alter the shuttling properties of the protein.…”

“…In fact, it has been shown that in AML patients with NPM1 mutations, which are always heterozygous, a small but detectable amount of wild-type NPM1 is always retained in nucleoli. 27 The rational for such approach is that, as NPM1 behaves as a hub protein in nucleoli, NPM1 nucleolar starvation would cause nucleolar stress and disruption of nucleolar structure [ Fig. 5(A)].…”