Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM

Abstract

Mutations of the gene for nucleophosmin (NPM1) are the most frequent genetic aberration in patients with acute myeloid leukemia (AML). The mechanism of leukemic transformation in this leukemia subtype is not fully understood, but aberrant cytoplasmic localization of mutated NPM (NPMmut) is widely considered as an important factor for leukemia manifestation. We analyzed the subcellular localization of three types of NPM with a C-terminal mutation (A, B and E). Genes for the individual NPM forms were fused with a gene for one of fluorescent protein variants in plasmids, which were transfected into three cell lines with different endogenous NPM expression. Subcellular localization of the fluorescent protein-labeled NPM was further correlated with the relative expression of all NPM forms. We confirmed a high cytoplasmic expression of NPMmutA and NPMmutB whereas a substantial fraction of NPMmutE was found to be localized in nucleoli. Moreover, we revealed that the localization of fluorescently labeled NPM is affected by the interaction between various forms of the protein.

Fig 1. NPM is localized in the cytoplasm of blast from AML patients with NPM mutation.

PBMC from AML patients with NPMwt (wt), NPMmutA (mut A) or NPMmutNm (mut Nm) were incubated with anti-NPM (clone 3F291) primary and AlexaFluor555 secondary antibodies (red). The nuclei were visualized with Hoechst 33342 (blue). Arrows indicate the cytoplasmic localization of NPM in AML blasts with NPMmut. The bars represent 10μm.

Fig 2. Subcellular distribution of mutated NPM depends on mutation type.

(a) eGFP fluorescence from HEK-293T cells transfected with eGFP plasmid (GFP), eGFP_NPMwt (wt), eGFP_NPMmutA (mutA), eGFP_NPMmutB (mutB) or eGFP_NPMmutE (mutE) showing various subcellular distribution of individual NPM variants. The bars represent 20μm. (b) fraction of transfected cells displaying eGFP_NPMmutA (or E) signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bars). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between mutA and mutE obtained from two-way ANOVA test was P < 0.001 (***). (c) immunoblot of lysates from HEK-293T cells transfected with individual NPM variants. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Densitometric evaluation of NPM exo/endo level and the ratio of NPMexo/endo expression vs the transfection efficiency (20%, 15%, 13,9% resp. 17,8% for wt, mutA, mutB resp. mutE) are indicated for the individual cell lines.

Fig 3. Interaction between wild-type and mutant affects localization of individual forms of NPM.

(a) eGFP (green) and mRFP1 (red) fluorescence from HEK-293T cells co-transfected with mRFP1_NPMwt and eGFP_NPMmutA (mutA) or eGFP_NPMmutE (mutE). The bars represent 20 μm. (b) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). GFP_mut denotes the signal from cells transfected with eGFP_NPMmut only, +RFP_wt denotes eGFP signal from cells co-transfected with eGFP_NPMmut and mRFP1_NPMwt. The error bars in the graph represent ±SD of at least 3 independent experiments. (c) fraction of transfected cells displaying mRFP1_NPMwt signal from the cytoplasm: wt—cells transfected only with RFP_NPMwt, wt+mutA (or E)–cells co-transfected with RFP_NPMwt and GFP_NPMmutA (or E). The error bars in the graph represent ±SD of 5 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***).

Fig 4. Localization of exogenous NPMmutA depends on endogenous NPM level.

(a) eGFP fluorescence from HEK-293T (1), NIH-3T3 (2) or HeLa (3) cells transfected with eGFP_NPMmutA showing its various subcellular distribution in individual cell lines. The bars represent 20μm. (b) immunoblot of lysates from various cell lines indicates different endogenous NPM expression. β-Actin represents the loading control. Densitometric evaluation of NPM/β-Actin ratio is indicated for individual cell lines. (c) fraction of transfected cells displaying eGFP_NPM signal only from the cytoplasm (white bars), from the cytoplasm and nucleoli (grey bars) or only from nucleoli (black bar). The error bars in the graph represent ±SD of at least 3 independent experiments. Statistical significance degree of difference between the samples: P < 0.01 (**), P < 0.001 (***). (d) immunoblot of lysates from various cell lines transfected with NPMmutA indicates different expression of transfected eGFP_NPM. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. Relative ratio of NPM exo/endo expression is indicated for the individual cell lines. Two-fold concentrations of primary and secondary antibodies had to be used to detect exogenous NPM expression in all lines. Therefore, absolute evaluation of the NPM exo/endo expression needs correction for the exo/endo NPM ratio calculated in Fig 2c.

Fig 5. Formation of heterooligomers between eGFP_NPM and endogenous NPM was confirmed by GFP-precipitation.

(a) Representative immunoblots of lysates and GFP-precipitates from the cells transfected with individual NPM variants. U: untransfected cells, wt: eGFP_NPMwt, mutA: eGFP_NPMmutA, mutE: eGFP_NPMmutE. Anti-NPM antibody clone NA24 was used to detect the overall NPM expression (i.e. both the NPMwt and NPMmut), the clone E3 was used to detect NPMwt only. GFP-NPM (exogenous) is detected at 64 kDa, the endogenous NPM at 37 kDa. β-Actin represents the loading control. (b) The ratio between endogenous (NPM) and exogenous (NPM-GFP) expression in GFP-precipitates and lysates from cells transfected with eGFP_NPMwt. The membrane from 30 to 100 kDa was incubated with anti-NPM clone NA24.