Understanding the specificity and regulation of NCK adaptor proteins

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The response of cells to extracellular stimuli is mediated by signalling pathways that act downstream of membrane bound receptors, such as the tyrosine kinase receptors (RTKs). These pathways are commonly organized through inducible protein-protein interactions and have been highly conserved throughout evolution. In particular, signalling pathways downstream of RTKs are essential for developmental patterning, organogenesis and homeostasis. Signals from RTKs are often relayed through adaptor proteins, which serve as hubs to recruit appropriate target proteins and guide signals to specific cellular pathways. They allow a relatively small number of receptor-ligand combinations to achieve a large range of outcomes depending on cell type and environment, by recruiting various combinations of regulatory proteins. The main goal is this research program is to decipher how normal cells use these adaptor proteins to coordinate specific responses from a given extracellular cue. In the case of RTKs, this is exemplified by adaptors composed exclusively of SH2 (Src Homology 2) and SH3 protein interaction domains, such as NCK1 and NCK2. NCK1/2 adaptors mediate the interaction between phosphorylated tyrosine (pTyr) containing proteins (via their single SH2) and effectors that contain poly-proline motifs (via one of their 3 SH3s). Little is known about NCK1 and NCK2 specificity as both adaptors are generally considered to be indistinguishable. In the last 5 years, we have identified several phosphorylated residues on NCK1/2, including an evolutionary-conserved Tyr within NCK1/2 SH3 domains that is phosphorylated by RTKs. We have further demonstrated that this Tyr phosphorylation abolished NCK1/2 protein interactions with SH3 targets, thus highlighting a new mode of regulation for NCK1/2. We have also shown that NCK1 and NCK2 signalling networks contain both common and unique components. We have identified NCK2-specific interaction partners that regulate processes such as cytokinesis, and further demonstrated an implication for NCK2, but not NCK1, in this process. We now seek to pursue this research program with the following aims: Aim 1- To define how NCK1/2 adaptors achieve specificity towards their targets, using NCK2 involvement in cytokinesis as a model; Aim 2- To determine how NCK1/2 adaptor proteins are regulated by phosphorylation and to explore how this impacts their functional signalling networks. We assert that the identification and characterization of NCK1- and NCK2-specific targets will be instrumental to understanding how adaptor proteins that appear identical nevertheless achieve specific functions in multiple aspects of cellular organization. Moreover, our studies will shed new light on SH2 and SH3 domains as protein interaction modules that mediate assembly of signalling complexes and as such, control the coordination of the cellular response to external signals.