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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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pone.0175175.g005: 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.

Mentions: A good correlation between the fraction of cells with cytoplasmic-only NPMmutA localization and the ratio of exogenous vs. endogenous NPM expression was observed. We suggest that heterodimers are formed not only between the fluorescently labeled NPM forms but also between the recombinant and the endogenous protein. This suggestion was further confirmed by eGFP-precipitation from lysates of transfected cells of HEK-293T and HeLa cell lines using GFP-Trap nanobeads (Fig 5a, S3 Fig). NPM expression was examined by two anti-NPM antibodies. The anti-NPM clone NA24 is directed to recognize the N-terminus of the human NPM and it is thus able to detect the overall NPM, i.e. both the NPMwt and NPMmut. The anti-NPM clone E3 is specific for an epitope at the C-terminus (aa 253–294) of NPMwt and it should hardly recognize the NPMmut. Indeed, whereas the clone NA24 detected GFP-NPM signal from all lysates of transfected cells, the clone E3 generated a signal only from samples transfected with eGFP-NPMwt. On the contrary, both clones equally detected the endogenous NPM in all precipitates containing any type of eGFP_NPM but not in precipitates from untransfected cells. Despite its relatively low expression in HeLa cells, eGFP_NPM effectively co-precipitated the endogenous NPM also in this cell line. Moreover, a higher ratio of the co-precipitated NPMwt vs. the precipitated eGFP_NPM corresponds to higher expression of the endogenous NPM in HeLa cells (Fig 5b, S3 Fig). For both cell lines, the amount of co-precipitated NPM was substantially higher in the samples from eGFP_NPMwt-transfected cells than in the samples transfected with eGFP_NPMmut.


Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM
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.
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Related In: Results  -  Collection

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pone.0175175.g005: 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.
Mentions: A good correlation between the fraction of cells with cytoplasmic-only NPMmutA localization and the ratio of exogenous vs. endogenous NPM expression was observed. We suggest that heterodimers are formed not only between the fluorescently labeled NPM forms but also between the recombinant and the endogenous protein. This suggestion was further confirmed by eGFP-precipitation from lysates of transfected cells of HEK-293T and HeLa cell lines using GFP-Trap nanobeads (Fig 5a, S3 Fig). NPM expression was examined by two anti-NPM antibodies. The anti-NPM clone NA24 is directed to recognize the N-terminus of the human NPM and it is thus able to detect the overall NPM, i.e. both the NPMwt and NPMmut. The anti-NPM clone E3 is specific for an epitope at the C-terminus (aa 253–294) of NPMwt and it should hardly recognize the NPMmut. Indeed, whereas the clone NA24 detected GFP-NPM signal from all lysates of transfected cells, the clone E3 generated a signal only from samples transfected with eGFP-NPMwt. On the contrary, both clones equally detected the endogenous NPM in all precipitates containing any type of eGFP_NPM but not in precipitates from untransfected cells. Despite its relatively low expression in HeLa cells, eGFP_NPM effectively co-precipitated the endogenous NPM also in this cell line. Moreover, a higher ratio of the co-precipitated NPMwt vs. the precipitated eGFP_NPM corresponds to higher expression of the endogenous NPM in HeLa cells (Fig 5b, S3 Fig). For both cell lines, the amount of co-precipitated NPM was substantially higher in the samples from eGFP_NPMwt-transfected cells than in the samples transfected with eGFP_NPMmut.

View Article: PubMed Central - PubMed

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.

No MeSH data available.