Étude de la spécificité fonctionnelle des protéines adaptatrices NCK1 et NCK2
|Abstract:||Signals from cell surface receptors are often relayed via adaptor proteins that can serve as hubs to recruit appropriate target signaling molecules and guide signals along specific pathways. Among these, adaptor proteins NCK1 (Non-Catalytic region of tyrosine Kinase 1) and NCK2 have functions that are often considered redundant and/or indistinguishable. The main goal of my work was to demonstrate that NCK1 and NCK2 are not fully redundant and may each display functional specificity. To achieve this, I delineated NCK1-and NCK2-specific signalling networks, identified for each unique target, then characterized what generates this specificity and obtained the function of these interactions. First, to identify the complement of interaction partners for NCK1 and NCK2, I used two unbiased mass spectrometry (MS)-based approaches: (i) epitope-tagged protein affinity purification (AP) followed by MS analysis and (ii) in vivo proximity labelling (BioID). The combination of these two approaches allowed me to identify more than one hundred specific interactions for each NCK. Bioinformatics analyzes based on the specific partners identified in MS enabled me to highlight that NCK2 was more specifically involved in the regulation of the actin cytoskeleton organization, structure essential for cell division and cytokinesis. By simultaneously comparing mouse embryo fibroblasts (MEF) depleted either for NCK1 or NCK2, I noticed that Nck2-/-, but not Nck1-/-cells are multi-nucleated and display extended protrusions reminiscent of intercellular bridges, which correlate with an extended time spent in cytokinesis as well as a failure of a significant proportion of cells to complete abscission. Further analysis of this phenotype revealed that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of the Polo-like kinase 1 (PLK1), Epithelial cell transforming 2 (ECT2) and Aurora B (AURKB) proteins. Moreover, I showed that NCK2 function during cytokinesis requires its SH2 domain. Second, to underline the molecular mechanism of specific protein complex formation, I selected based on my MS results 27 partners to confirm by an orthogonal method their respective interactions with NCK1 and/or NCK2. By using in vitro binding assays, I was able to determine that several proteins including Plakophilin 4(PKP4), a key regulator of the cytokinesis process, were able to bind directly and specifically to NCK2. Through various in vitro experiments, I was able to determine that NCK2 binds the N-terminal and central portions of PKP4 through its SH2 domain and that the specificity of PKP4 toward NCK2 does not appear to result from the intrinsic properties of its SH2 alone. This association seems to result from the combination of some or all of the properties of the individual domains and inter-regions constituting the NCK1/2 proteins. In conclusion, despite what is generally accepted, I showed that both NCK1 and NCK2 may form specific protein complexes, thus reflecting the functional specificity of these two adaptor proteins. I further demonstrated that NCK1 and NCK2 are not completely redundant. I also shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division. Finally, my in vitro experiments provide an explanation for the specificity mechanism of NCK1/2 adaptor proteins by suggesting that their specificity come from the combination of the properties of their respective domains and/or interdomain regions.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||11 July 2019|
|Collection:||Thèses et mémoires|
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