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International audience; Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Signals from cell surface receptors are often relayed via adaptor proteins. NCK1 and NCK2 are Src-Homology (SH) 2 and 3 domain adaptors that regulate processes requiring a remodeling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggests that NCK1 and NCK2 are functionally redundant, although recent reports support the idea of unique functions for NCK1 and NCK2. We sought to examine this question further by delineating NCK1- and NCK2-specific signaling networks. We used both affinity purification-mass spectrometry and BioID proximity labeling to identify NCK1/2 signaling networks comprised of 98 proteins. Strikingly, we found 30 proteins restricted to NCK1 and 28 proteins specifically associated with NCK2, suggesting differences in their function. We report that Nck2(−/−), but not Nck1(−/−) mouse embryo fibroblasts (MEFs) are multinucleated 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. Our data also show that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of AURKB, PLK1 and ECT2. Finally, we show that NCK2 function during cytokinesis requires its SH2 domain. Taken together, our data delineate the first high-confidence interactome for NCK1/2 adaptors and highlight several proteins specifically associated with either protein. Thus, contrary to what is generally accepted, we demonstrate that NCK1 and NCK2 are not completely redundant, and shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division.

Protein phosphorylation often regulates interactions between components of signalling networks. Its tight regulation by opposing actions of kinases and phosphatases contribute to making this modification key in controlling the dynamic architecture of phosphorylation-dependent networks. The introduction in cell signalling research of long-standing targeted proteomics approaches such as selected reaction monitoring (SRM) combined with the development of state-of-the-art methods such as parallel reaction monitoring (PRM) and data-independent analysis (DIA), have allowed delineating temporal quantitative profiles of interactions networks. This review summarizes how targeted proteomics analyses have recently contributed to our comprehension of phosphorylation-dependent protein interaction networks and proposes how these could be applied to address clinical problems. BIOLOGICAL SIGNIFICANCE: Intracellular communication and information processing are performed by context-specific protein interaction networks that are often regulated by protein phosphorylation. Quantification of dynamic changes within these signalling networks is of critical importance to understand cell biology in normal and disease states, but remains challenging. Targeted proteomics approaches have contributed greatly to our understanding of these phosphorylation-dependent signalling networks.

Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.