Understanding the specificity and regulation of NCK adaptor proteins

细胞对细胞外刺激的反应是通过作用于膜结合受体下游的信号通路来调节的，如酪氨酸激酶受体(RTK)。这些途径通常是通过可诱导的蛋白质-蛋白质相互作用来组织的，并且在整个进化过程中高度保守。特别是，RTK下游的信号通路对于发育模式、器官发生和动态平衡是必不可少的。RTK的信号通常通过接头蛋白传递，接头蛋白作为枢纽招募适当的靶蛋白，并将信号引导到特定的细胞通路。它们允许数量相对较少的受体-配体组合通过招募各种调节蛋白组合来实现取决于细胞类型和环境的大范围结果。这个研究计划的主要目标是破译正常细胞如何使用这些接头蛋白来协调给定细胞外线索的特定反应。在RTK的情况下，这通过完全由SH2(Src同源2)和SH3蛋白相互作用结构域组成的适配器来例证，例如NCK1和NCK2。NCK1/2接头介导含有磷酸化酪氨酸(PTyr)的蛋白质(通过其单一的SH2)与含有多-脯氨酸基序的效应器(通过其3个SH3中的一个)之间的相互作用。人们对NCK1和NCK2的特异性知之甚少，因为这两个接头通常被认为是不可区分的。在过去的5年里，我们已经在NCK1/2上发现了几个磷酸化残基，包括一个进化保守的被RTKs磷酸化的NCK1/2SH3结构域中的Tyr。我们进一步证明了这种Tyr磷酸化取消了NCK1/2蛋白与SH3靶标的相互作用，从而突出了NCK1/2的一种新的调控模式。我们还证明了NCK1和NCK2信号网络既包含共同的组件，也包含独特的组件。我们已经确定了调控胞质分裂等过程的NCK2特异性相互作用伙伴，并进一步证明了NCK2在这一过程中的含义，但不是NCK1。目标1-以NCK2参与胞质分裂为模型，确定NCK1/2接头蛋白是如何实现靶点特异性的；目标2-确定NCK1/2接头蛋白如何通过磷酸化来调节，并探索这如何影响其功能信号网络。我们断言，NCK1和NCK2特异性靶标的识别和表征将有助于理解看似相同的接头蛋白如何在细胞组织的多个方面实现特定功能。此外，我们的研究将为SH2和SH3结构域作为蛋白质相互作用模块提供新的线索，这些模块介导信号复合体的组装，从而控制细胞对外部信号的反应的协调。

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.