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.

Conformational stabilization as a strategy to prevent nucleophosmin mislocalization in leukemia

“…Similar results were obtained with the HEK293T cell line (not shown). The observed behavior agrees with previously published results using ectopically expressed fluorescent protein-NPM constructs 27 , 28 , and reproduces the partially cytoplasmic localization of NPM in blasts of AML patients 29 , suggesting that this system is a valid model of the localization dynamics of NPM in AML. Additionally, and only in cells expressing mutant variants of YFP-NPM, we observed the presence of bright fluorescent dots, which we attributed to protein aggregation, probably due to the defective folding of the mutant proteins (Fig.…”

“…On the other hand, those cells expressing low amounts of mutant (A or E) YFP-NPM displayed less aggregates and relatively more nucleolar NPM staining. This could be due to the influence of endogenous, wild type NPM, expected to form mixed oligomers with the mutant protein 28 . Nevertheless, the aggregation cannot exclusively be ascribed to excessive overexpression of the constructs, since the exogenous:endogenous NPM ratio normalized by the transfection efficiency did not correlate with the presence of aggregates.…”

The acute myeloid leukemia‐associated Nucleophosmin 1 gene mutations dictate amyloidogenicity of the C‐terminal domain

“…The cytoplasmic translocation impairs NPM1 functions and determines the binding with other cytoplasmic proteins and the enhancement of OCI‐AML3 cells proliferation is likely due to a downregulatory effect on the ARF tumor‐suppressor gene and to an enhancer effect on the c‐MYC oncogene . Furthermore, mutated proteins present peculiar features in their cellular localization, also due to their hetero‐oligomerization with wt form: the ability of NPM1 to form oligomers is never disrupted by any type of C‐terminal mutation but seems to be generally lowered, irrespective of the mutation type . The combination of haploinsufficiency and the gain‐of‐function of NPMc+ contribute to the pathogenesis of AML .…”

Differential expression of NPM, GSTA3, and GNMT in mouse liver following long-term in vivo irradiation by means of uranium tailings

“…NPM is a multifunctional oligomeric phosphoprotein and is encoded by the NPM1 gene [ 53 , 54 ]. NPM1mA is a mutant form of the NPM1 gene and is the most common genetic abnormality in patients with acute myeloid leukemia (AML) [ 55 , 56 ]. NPM1mA functions as an oncogene and is present in ∼30% of patients with AML [ 57 ].…”