Identification of a novel nucleophosmin‐interaction motif in the tumor suppressor p14arf

Abstract

The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.

Nucleophosmin in Its Interaction with Ligands

“…Importantly, a novel region on p14ARF that interacts with NPM1 N-terminal domain was identified in the C-terminal tract of p14ARF. This region is also rich in arginine residues and predicted to constitute the p14ARF NoLS [113,114]. These findings were consistent with the ability of NPM1 to recognize putative NoLS sequences in its interactors (see below) [33,115].…”

“…Given the numerous proteins bound by NPM1, inhibition of these interactions might affect its role as a "hub" protein within the nucleoli and result toxic [15,161]. As already outlined in the previous section, a large surface in the N-terminal domain of NPM1 serves as a docking site for proteins carrying a NoLS rich in positively charged residues [26,113,115]. In the past years, NPM1 has been identified as one of the targets of the synthetic pseudopeptide NucAnt N6L (hereby N6L), a pro-apoptotic compound able to exert antiproliferative activity and to inhibit tumor growth also in vivo, that has already completed Phase I/IIa clinical trials for different solid tumors [162][163][164].…”

“…A complex equilibrium between folded pentamers and partly or totally unfolded monomers, mainly influenced by the phosphorylation status of several residues in the core domain, was envisaged [ 26 , 30 ]. More recently, these studies were integrated by analyzing the interaction in the context of full-length human p14ARF [ 113 ]. It was shown, by NMR, that p14ARF is completely unfolded and tends to associate, through its N-terminal end, in grossly insoluble aggregates.…”

The nucleolus, an ally, and an enemy of cancer cells

“…Moreover, NPM is induced by the Ras oncogene merely in the ARF-deprived cells which additionally proves ARF anticancer function (Itahana et al 2003 ; Pandit and Gartel 2015 ). Besides, the activity of ARF itself depends on NPM, because its nucleolar localization is conditioned by the sequestration with NPM, provided by specific interaction between these two proteins (Luchinat et al 2018 ), which limits its extranucleolar functioning (Korgaonkar et al 2005 ). Moreover, it has been proposed that this interaction creates specific microenvironment facilitating nucleolar stress response (Mitrea and Kriwacki 2018 ).…”

Emerging evidence of the molecular landscape specific for hematogenous metastasis from gastric cancer

“…Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein involved in numerous cellular processes, including centrosome duplication, histone assembly, protein chaperoning and cell proliferation[ 113 , 114 ]. NPM1 downregulates the tumor suppressor cyclin dependent kinase inhibitor 2A in the nucleolus and has inhibitory effects by activating transcription factor 5 (ATF5) and abrogating ATF5-induced G(2)/M cell cycle blockade[ 115 , 116 ]. Some studies revealed that positive expression of NPM1 in GC tissue was associated with poor prognosis in postoperative GC patients.…”