CONICET | Buscador de Institutos y Recursos Humanos

Advances in quantitative mass spectrometry-based proteomics now enables the system-wide characterization of signaling events at the level of post-translational modifications, protein-protein interactions and changes in protein expression. The 14-3-3 proteins interact with more than 800 different proteins, in part as the result of their specific phospho-serine/phospho-threonine binding activity (RSXpS/TXP, RXXXpS/TXP and pS/T-X(1-2)-COOH). The family is composed by 2 paralogs in yeast, 7 in mammals, and up to 15 in plants. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. 14-3-3 can hide intrinsic localization motifs, prevent molecular interactions and/or modulate the accessibility of a target protein to modifying enzymes such as kinases, phosphatases or proteases. The extraordinarily high sequence conservation between 14-3-3 protein isoforms poses a significant technological challenge to researchers working with this family. A systems-level approach is necessary to map 14-3-3 networks components and to understand their functions. We used different databases to create a PPI (protein-protein interaction) network for 14-3-3 signaling in human cells. We also added kinases and their substrates published in the HPRD database for human cells, including the information about the phosphorylation- and Lys acetylation sites. Finally we transformed this unidirectional network of ~5000 nodes in a directed one, obtaining a complete representation at high resolution of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each isoform are statistically different (Jaccard index < 0.25) and built by different set of 3-nodes motifs (p < 0.005), with different structural stability. A feed-forward loop motif (# 7, SSS=1) is present in gamma, zeta and eta networks. This motif has been detected within the transcription-regulation networks of E. coli and S. cerevisiae. At the level of signal transduction networks, this motif could represent the scaffold function, where a protein (in this case 14-3-3) facilitates the interaction between two other proteins (one of them regulates the other one). Another feature that shows differences between each isoform specific network is the intrinsic disorder content (p = 2.044e-09 Krustal-Walis test), promoting distinct levels of wired interactome. This difference in the percentage of disorder is reflected in the size, number and co-appearance of domains and domains clubs in each partner of 14-3-3 network isoforms, suggesting their participation in different signaling pathways. It was remarkable to found that Tyr was the most phosphorylable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains suggest that epsilon could be involved in growth factors receptors signaling pathways. Finally, we found that within zetas network, the number of acetylated partners is significantly higher (Fisher exact test) compared with each of the other isoforms, with p values from 1.65e-10 for sigma, the less similar, to 0.0024 for gamma, the most similar to zeta isoform. The number of acetylated Lys is not proportional to the domain number (or number of amino acids in domains). In the case of zeta isoform, the domains of its partners contain more modified Lys than all 14-3-3 paralogs. Also, an analysis of the subcellular localization of those zeta partners that are acetylated (48%) shows that 42% are mainly nuclear, containing the 60% of all Nuclear Localization Signals present in partners of this isoform (p = 1.288e-06, Fisher exact test). The Lys acetylation correlates with pTyr but not with pSer or pThr, suggesting a crosstalk between these two kinds of PTM. Our analysis also shows a clear subfunctionalization in members of the 14-3-3 family by differential PTMs.

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