Signal Transduction by Tyrosine Phosphorylation

描述（由申请人提供）：信号事件的适当定位和动力学对于实现细胞内信号的效率、特异性和多样性至关重要。我们计划研究信号如何传递到细胞中的肌动蛋白细胞骨架，以指示响应酪氨酸激酶介导的信号和粘附到细胞外基质（ECM）蛋白的局部组装和重塑。为了帮助这些研究，我们还建议开发基于基因编码的fret激酶活性报告器（生物传感器）来研究单个活细胞内蛋白激酶活性的时空模式。具体来说，我们计划研究c-Abl酪氨酸激酶调节肌动蛋白细胞骨架组装和促进细胞表面丝状伪足形成的途径。我们将测试在纤维连接蛋白上扩散的成纤维细胞中，Dokl接头被c-Abl磷酸化的模型，并导致Nck SH2/3接头蛋白的募集。我们将研究Nck如何传递与Rho家族无关的信号来启动f -肌动蛋白组装。我们将使用Abl激酶生物传感器和gfp融合蛋白，精确地确定这一途径在扩散细胞中何时何地被激活，以确定这如何引发含有f -肌动蛋白的丝状足的形成。此外，我们将分析EphA2受体酪氨酸激酶信号通路，这些信号通路在成纤维细胞中被激活，在ephrin A1配体包被的表面上扩散，并导致整合素的激活和肌动蛋白细胞骨架的组装，重点关注Rap 1a和Rac被p 130Cas/Crk复合物激活的机制，Src/FAK复合物的信号传导，以及在扩张性成纤维细胞中观察到的Rac/Cdc42活化与f -肌动蛋白细胞骨架的特征性重组的耦合。我们将使用生物传感器和gfp融合蛋白确定这些通路在扩散细胞中的激活位置和时间，并将我们所学到的应用于神经元细胞中的ephrin/Eph信号传导。最后，我们建议开发多种基于fret的生物传感器蛋白，专门报告参与DNA损伤反应的MAP激酶的ERK， JNK和p38家族成员的活性，以及ATM/ATR PIKK家族激酶的活性，并将这些报告者用于培养活细胞和线虫和斑马鱼发育过程中的研究，以确定这些蛋白激酶在何时何地被激活。总体目标是在高分辨率下研究细胞内蛋白质磷酸化介导的信号转导事件的时空动态，并定义它如何在细胞中产生局部输出，例如肌动蛋白细胞骨架的组织，以响应外部刺激。

DESCRIPTION (provided by applicant): The proper localization and dynamics of signaling events are essential for achieving the efficiency, specificity and diversity of intracellular signaling. We plan to study how signals are transmitted to the actin cytoskeleton in cells to dictate local assembly and remodeling in response to tyrosine kinase mediated signaling and following adhesion to extracellular matrix (ECM) proteins. To aid in these studies, we also propose to develop genetically encoded FRET-based kinase activity reporters (biosensors) to study the spatiotemporal patterns of protein kinase activity within single living cells. Specifically, we plan to investigate the pathways used by the c-Abl tyrosine kinase to regulate assembly of the actin cytoskeleton and promote formation of surface filopodia in cells spreading on ECM. We will test the model that the Dokl adaptor is phosphorylated by c-Abl in fibroblasts spreading on fibronectin, and that this leads to recruitment of the Nck SH2/3 adaptor protein. We will study how Nck transmits a Rho family-independent signal to initiate F-actin assembly. We will determine precisely where and when this pathway is activated in spreading cells, using Abl kinase biosensors and GFP-fusion proteins, to establish how this elicits formation of F-actin containing filopodia. In addition, we will analyze the EphA2 receptor tyrosine kinase signaling pathways that are activated in fibroblasts spreading on an ephrin A1 ligand coated surface, and lead to activation of integrins and assembly of the actin cytoskeleton, focusing on the mechanisms of activation of Rap 1a and Rac by p 130Cas/Crk complexes, signaling by Src/FAK complexes, and the coupling of Rac/Cdc42 activation to the characteristic reorganization of the F-actin cytoskeleton observed in spreading fibroblasts. We will determine where and when these pathways are activated in spreading cells using biosensors and GFP-fusion proteins, and apply what we learn to ephrin/Eph signaling in neuronal cells. Finally, we propose to develop multiple FRET-based biosensor proteins that specifically report the activities of members of the ERK, JNK and p38 families of MAP kinases, and of ATM/ATR PIKK family kinases involved in the DNA damage response, and employ these reporters for studies in living cells in culture and during C. elegans and zebrafish development to determine where and when these protein kinases are activated. The overall goal is to investigate at high resolution the spatial and temporal dynamics of protein phosphorylation-mediated signal transduction events within the cell, and define how this generates localized outputs in the cell, such as the organization of the actin cytoskeleton, in response to external stimuli.