Signal Transduction by Tyrosine Phosphorylation

描述（由申请人提供）：信号传导事件的适当定位和动力学对于实现细胞内信号传导的效率、特异性和多样性至关重要。我们计划研究信号是如何传递到细胞中的肌动蛋白细胞骨架，以决定局部组装和重塑，以响应酪氨酸激酶介导的信号传导和粘附到细胞外基质（ECM）蛋白。为了帮助在这些研究中，我们还建议开发基因编码的FRET为基础的激酶活性报告（生物传感器），研究蛋白激酶活性在单个活细胞内的时空模式。具体来说，我们计划调查的c-Abl酪氨酸激酶调节组装的肌动蛋白细胞骨架和促进形成的表面丝状伪足在细胞外基质上传播的途径。我们将测试Dokl衔接子在纤连蛋白上铺展的成纤维细胞中被c-Abl磷酸化的模型，并且这导致Nck SH 2/3衔接子蛋白的募集。我们将研究Nck如何传递独立于Rho家族的信号来启动F-actin组装。我们将使用Abl激酶生物传感器和GFP融合蛋白，精确地确定在扩展细胞中何时何地激活这一途径，以确定这是如何促进含有丝状伪足的F-肌动蛋白的形成的。此外，我们将分析EphA 2受体酪氨酸激酶信号通路，该通路在成纤维细胞中在ephrin A1配体包被的表面上铺展时被激活，并导致整合素的激活和肌动蛋白细胞骨架的组装，重点是通过p130 Cas/Crk复合物激活Rap 1a和Rac，通过Src/FAK复合物信号传导，以及Rac/Cdc 42活化与在扩散成纤维细胞中观察到的F-肌动蛋白细胞骨架的特征性重组的偶联。我们将使用生物传感器和GFP融合蛋白确定这些途径在扩散细胞中何时何地被激活，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，我们建议开发多个FRET为基础的生物传感器蛋白，具体报告的活动的ERK，JNK和p38家族的MAP激酶，和ATM/ATR PIKK家族激酶参与DNA损伤反应的成员，并采用这些报告在活细胞培养和在C。线虫和斑马鱼的发育，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，并确定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.