Abstract
Simple SummaryNucleophosmin (NPM) is one of the most abundant nucleolar proteins and its mutations frequently occur in acute myeloid leukemia (AML). The mutations cause aberrant cytoplasmic localization of mutated protein (NPMmut) and often mediate dislocation of NPM interaction partners. Tumor suppressor p53 is known to interact with NPM in response to genotoxic stress and its cytoplasmic localization is an unfavorable prognostic factor in cancers. This study aims to characterize the NPM-p53 interaction and to elucidate the effect of the NPM mutations on p53 localization and expression in live cells. In addition, the cellular dynamics of NPMmut and p53 after treatment with nuclear export inhibitor Selinexor is described and the mechanism of the Selinexor action proposed. Our results contribute to a better understanding of the oncogenic potential of NPM mutations.Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.
Highlights
Nucleolus is a subnuclear compartment with multiple cellular roles, including cellular stress sensing [1]
In the second part of this work, we address p53 interactions and trafficking in HEK-293T cells and leukemia cell lines treated with Selinexor in order to elucidate mechanism of p53 and NPMmut co-translocation in live cells
As NPM forms complex with p53 [3,4] and we found this complex in intact cells, we expect that p53 and NPMmut are simultaneously co-exported out of the nucleus in the p53/NPMmut complex
Summary
Nucleolus is a subnuclear compartment with multiple cellular roles, including cellular stress sensing [1]. During the stress response nucleoli become more condensed and nucleolar proteins partially translocate to the nucleoplasm. Nucleolar phosphoproteins, such as nucleophosmin (NPM) or nucleolin (NCL), participate in the stress response via complex interaction network [2]. The translocated proteins can stabilize tumor suppressor p53 either by direct interaction [3,4,5,6] or by interaction with proteins regulating intracellular p53 levels [7]. NPM is an abundant nucleolar phosphoprotein with numerous functions in important cellular processes, including the p53-dependent response to genotoxic stress [1,8]. NPM interacts with p53 as well as with other tumor suppressors, such as pRb or p14Arf [2,9,10]. While the interaction with p53 occurs via region close to the C-terminus of NPM [3], the N-terminal NPM region is responsible for NPM-oligomerization [11,12]
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