Structural basis for the recognition by 14-3-3 proteins of a conditional binding site within the oligomerization domain of human nucleophosmin

Kapitonova, Anna A.; Tugaeva, Kristina V.; Varfolomeeva, Larisa A.; Boyko, K.М.; Cooley, Richard B.; Sluchanko, Nikolai N.

doi:10.1016/j.bbrc.2022.08.047

Cited by 4 publications

(4 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Ser48-containing motif is the best 14-3-3-binding candidate in human NPM1 (Table 1); its phosphorylation was shown to trigger 14-3-3 binding to the surrounding peptide fragment [22]. T78E, and especially S112E, NPM1 versions containing partially phosphorylated Ser48 do recruit 14-3-3γ, but still the complex formation was limited by phosphorylation efficiency (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The binding curves were approximated by the quadratic equation [63]. L. Structural model showing the absence of steric clashes and stabilizing interactions between FSC and the pS48 phosphopeptide based on the 14-3-3ζ/NPM1-pS48 peptide structure (PDB 8ah2 [22]). M. Structural model showing the canonical FSC-stabilized interaction between 14-3-3σ and the pS293 phosphopeptide (PDB 7obg and 7obh [23]) with the contacts formed between the peptide's COOH group and 14-3-3 and FSC.…”

Section: Chemicals Molecular Cloning Protein Expression and Purificat...mentioning

confidence: 99%

“…Reasons for this discrepancy are not clear but it could be because NPM1/14-3-3 interactions depend on the specific pattern of NPM1 phosphorylation across multiple sites. Intriguingly, recent crystal structures have provided snapshots of 14-3-3 interaction with two NPM1 phospho-fragments, around pSer48 [22] and pSer293 [23]. NPM1 phosphorylation at Ser48 by prosurvival kinase Akt led to NPM1 pentamer disassembly and exit from the nucleolus [7], and so not surprisingly NPM1 phosphorylated at Ser48 is prevalent in human tumors [7].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

Kapitonova,

Perfilova,

Cooley

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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 phospho-sites in NPM1 reside within signal sequences, this work highlights a key mechanism of NPM1 regulation by which NPM1 phosphorylation promotes 14-3-3 binding to control nucleocytoplasmic shuttling. 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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Chemicals Molecular Cloning Protein Expression and Purificat...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

Kapitonova,

Perfilova,

Cooley

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…His-tagged full-length human 14-3-3ε was obtained in previous work (Tugaeva et al ., 2020). Lambda phosphatase was obtained as a recombinant protein with the N-terminal His 6 -tag cleavable by human rhinovirus 3C protease in previous work (Kapitonova et al ., 2024). The dephosphorylation reaction (20 µl) lasted 5 h at 20 °C and contained 1 mg/ml of purified plant 14-3-3 samples or pre-phosphorylated HSPB6 in 20 mM Tris-HCl pH 7.8 with 150 mM NaCl, 1.9 mM MnCl 2 , 5 mM dithiothreitol (DTT) and 1/10 diluted lambda phosphatase.…”

Section: Methodsmentioning

confidence: 99%

Multi-dimensional demarcation of phylogenetic groups of plant 14-3-3 isoforms using biochemical signatures

Sedlov,

Sluchanko

2024

Preprint

Self Cite

View full text Add to dashboard Cite

Interaction of dimeric 14-3-3 proteins with numerous phosphotargets regulates various physiological processes in plants, from flowering to transpiration and salt tolerance. Several genes express distinct 14-3-3 ‘isoforms’, particularly numerous in plants, but comparative studies of all 14-3-3 isoforms for a given organism have not been undertaken. Here we systematically investigated twelve 14-3-3 isoforms from the model plantArabidopsis thaliana, uniformly capable of homodimerization at high protein concentration. We unexpectedly discovered that, at physiological protein concentrations, four isoforms representing a seemingly more ancestral, epsilon phylogenetic group (iota, mu, omicron, epsilon) demonstrate an outstanding monomerization propensity and enhanced surface hydrophobicity, which is uncharacteristic for eight non-epsilon isoforms (omega, phi, chi, psi, upsilon, nu, kappa, lambda). Further analysis revealed that dramatically lowered thermodynamic stabilities entail aggregation of the epsilon-group isoforms at near-physiological temperatures and provoke their proteolytic degradation. Structure-inspired single mutations in 14-3-3 iota could rescue non-epsilon behavior, thereby pinpointing key positions responsible for the phylogenetic demarcation. Combining two major demarcating positions (namely, 27th and 51st in omega) and multi-dimensional differences in biochemical properties identified here, we developed a predictor strongly supporting categorization of abundant 14-3-3 isoforms widely across plant groups, from Eudicots to Monocots, Gymnosperms and Lycophytes. In particular, our approach fully recapitulates the phylogenetic epsilon/non-epsilon demarcation in Eudicots and supports the presence of isoforms of both types in more primitive plant groups such asSelaginella, thereby refining solely sequence-based analysis in evolutionarily distant species and providing novel insights into the evolutionary history of the epsilon phylogenetic group.SignificanceDespite over 30 years of research, systematic comparative studies on the regulatory plant 14-3-3 proteins have not been undertaken, making phylogenetic classification of numerous plant 14-3-3 isoforms in different species unreliable. Working on twelve purifiedArabidopsis14-3-3 isoforms, we have discovered a set of biochemical signatures that can be used to robustly and widely categorize epsilon and non-epsilon plant 14-3-3 isoforms, also identifying at least two amino acid positions responsible for such multi-dimensional demarcation.

show abstract

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein

Sluchanko,

Kapitonova,

Shulepko

et al. 2023

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

Structural basis for the recognition by 14-3-3 proteins of a conditional binding site within the oligomerization domain of human nucleophosmin

Cited by 4 publications

References 50 publications

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

Multi-dimensional demarcation of phylogenetic groups of plant 14-3-3 isoforms using biochemical signatures

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein

Contact Info

Product

Resources

About