Phosphorylation Code of Human Nucleophosmin Includes Four Cryptic Sites for Hierarchical Binding of 14-3-3 Proteins

• Published in: Journal of molecular biology Vol. 436; no. 12; p. 168592
• Main Authors: Kapitonova, Anna A., Perfilova, Kristina V., Cooley, Richard B., Sluchanko, Nikolai N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15.06.2024
• Subjects: 14-3-3 Proteins - chemistry; 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; Binding Sites; cryptic disorder; domain-motif interaction; fluorescence anisotropy; Humans; Nucleophosmin - chemistry; Nucleophosmin - genetics; Nucleophosmin - metabolism; oligomeric state transition; Phosphorylation; Protein Binding; Protein Multimerization; cryptic disorder; domain-motif interaction; oligomeric state transition; phosphorylation; fluorescence anisotropy
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2024.168592

[Display omitted] •NPM1 is phosphorylated by PKA at multiple sites phosphorylatable in vivo.•Phosphorylation drives NPM1 unfolding, monomerization and recruits 14-3-3.•Four NPM1 phosphoserines are recognized by all seven human 14-3-3 isoforms.•Binding of phosphomotifs is hierarchical, pS293 > pS48 > pS106 > pS260.•Fusicoccin selectively inhibits or stabilizes these 14-3-3/phosphomotif complexes. Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phosphosites in NPM1 reside within signal sequences, this work suggests a mechanism of NPM1 regulation by which NPM1 phosphorylation can promote 14-3-3 binding to affect NPM1 shuttling between cell compartments. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.