Abstract
The cytoplasmic mutant of nucleophosmin (NPMc) is found approximately in one-third of acute myeloid leukemia (AML) cases and is highly associated with normal karyotype. Whereas previous studies have focused on wtNPM in centrosome duplication, we further elucidate the role of NPM in the cell cycle by utilizing the increased cytoplasmic load of NPMc. Overexpression of NPMc causes increased phosphorylation of NPM on T199 and, to a lesser degree, S4. T199 phosphorylation is dependent on cdk2 but activators of cdk2 were not elevated. Upon inhibition of cdk2, NPMc-overexpressing cells demonstrate a greater G2/M phase arrest than wtNPM or GFP counterparts. However, the number of cells with 2 centrosomes did not increase concordantly. This suggests that the arrest was caused by a delay in centrosome duplication, most likely due to the inhibition of centrosome duplication caused by unphosphorylated NPMc. Overall, these results suggest that the phosphorylation of T199 is important in the mitotic progression of NPMc-expressing cells. This further supports the hypothesis that NPMc is associated with normal karyotypes in AML because the higher cytoplasmic load of NPM can better suppress centrosome overduplication which would otherwise result in unequal segregation of chromosomes during mitosis, leading to aneuploidy and other genomic instabilities.
Highlights
With the duplicated centrosomes during mitosis where it is phosphorylated by the mitotic cyclin B-cdk[1] complex[14] on T234 and T23715
In order to study the role of NPMcT199 phosphorylation in centrosome duplication, HEK293T cells were transduced to stably express green fluorescent protein (GFP), GFP tagged wtNPM or GFP-NPMc (NPMc)
This is consistent with the hypothesis that in G1/S/G2 phases when the nuclear envelope is still intact and NPM is phosphorylated to permit centrosome duplication, many more molecules of NPM need to be phosphorylated in NPMc expressing cells due to the increased load of NPM in the cytoplasm
Summary
With the duplicated centrosomes during mitosis where it is phosphorylated by the mitotic cyclin B-cdk[1] complex[14] on T234 and T23715. Mutation of T199 to non-phosphorylatable alanine prevents phosphorylation and centrosome duplication, increasing the number of cells with one centrosome and decreasing the number of cells with two centrosomes[16]. Without this phosphorylation, NPM associates on unduplicated centrosomes and after phosphorylation dissociates leaving duplicated centrosomes free of NPM17. Centrosome numbers did not change significantly which is in discord with the change in cell cycle profile. This desynchronization of the centrosome and DNA duplication cycles show that T199 phosphorylation is especially important for cell cycle progression when NPM is mislocalized in cytoplasm
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