Understanding the specificity and regulation of NCK adaptor proteins

细胞对细胞外刺激的反应由作用于膜结合受体（例如酪氨酸激酶受体（RTK））下游的信号传导途径介导。这些途径通常通过诱导蛋白质-蛋白质相互作用来组织，并且在整个进化过程中高度保守。特别是，RTK下游的信号传导途径是必不可少的发育模式，器官发生和稳态。来自RTK的信号通常通过衔接蛋白传递，衔接蛋白作为枢纽招募适当的靶蛋白并将信号引导至特定的细胞途径。它们允许相对少量的受体-配体组合，通过招募各种调节蛋白的组合，根据细胞类型和环境实现大范围的结果。这项研究计划的主要目标是破译正常细胞如何使用这些衔接蛋白来协调特定细胞外信号的特异性反应。在RTK的情况下，这通过仅由SH 2（Src同源物2）和SH 3蛋白相互作用结构域（例如NCK 1和NCK 2）组成的衔接子来例示。NCK 1/2衔接子介导含磷酸化酪氨酸（pTyr）的蛋白质（通过它们的单个SH 2）和含聚脯氨酸基序的效应子（通过它们的3个SH 3之一）之间的相互作用。关于NCK 1和NCK 2特异性知之甚少，因为这两种衔接子通常被认为是不可区分的。 在过去的5年里，我们已经鉴定了NCK 1/2上的几个磷酸化残基，包括NCK 1/2 SH 3结构域中被RTK磷酸化的进化保守Tyr。我们进一步证明了这种Tyr磷酸化消除了NCK 1/2蛋白与SH 3靶点的相互作用，从而突出了NCK 1/2的新调控模式。我们还表明，NCK 1和NCK 2信号网络包含共同和独特的组件。我们已经确定了NCK 2特异性的相互作用伙伴，调节过程，如胞质分裂，并进一步证明了NCK 2的影响，但不是NCK 1，在这个过程中。我们现在寻求进行这项研究计划，其目标如下： 目的1-以NCK 2参与胞质分裂为模型，确定NCK 1/2衔接子如何实现对其靶点的特异性; 目的2-确定NCK 1/2接头蛋白如何通过磷酸化调节，并探索这如何影响其功能信号网络。 我们断言，NCK 1和NCK 2特异性靶点的鉴定和表征将有助于理解看似相同的衔接蛋白如何在细胞组织的多个方面实现特定功能。此外，我们的研究将揭示新的光SH 2和SH 3域作为蛋白质相互作用模块，介导组装的信号复合物，因此，控制协调细胞对外部信号的反应。

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.