Src-mediated pathways regulating adherens junction assembly.

项目摘要 内皮屏障在黏附连接(AJs)水平上被调节，细胞-细胞黏附结构介导 通过跨膜蛋白VE-钙粘附素。目前的模型表明，酪氨酸激酶c-Src的功能是 内皮屏障负性调节因子通过VE的磷酸化刺激AJs的分解 钙粘附素。然而，我们的研究挑战了这一范式。我们发现，使用我们的 工程探针瞬时增强屏障功能并诱导形态不同的AJ形成 表现出渗透性降低的情况。我们的初步证据表明，Src诱导的增强 内皮屏障是通过调节小GTP酶Arf6的信号通路介导的。我们还发现， Tyr658/Tyr731上VE钙粘附素的磷酸化是Src增强屏障的关键。我们的研究也 显示Src促进与细胞外基质形成新的接触(局灶性粘连)并刺激 粘着斑蛋白p130Cas与VE钙粘附素的相互作用基于这些新发现，我们假设 通过Arf6和VE-cadherin的Src信号转导，以及对新的局部粘连的刺激，促进了 AJS导致内皮屏障增强。为了定义Src下游的每条路径的作用，我们 建议解决以下问题。1)我们将确定Arf6活性在Src刺激中的作用 AJs的形成，并确定Src介导的激活Arf6的途径。2)我们将定义服务提供商的角色 AJs中的信号转导及VE-cadherin上单个磷酸化位点在新AJs和AJs形成中的作用 内皮通透性增强。3)我们将确定局部粘连在Src介导的过程中的作用 血管内皮细胞屏障的增强和通过特异的局部黏附确定Src信号的作用 蛋白质。Src介导的信号转导的时间和位置对AJ的调控至关重要。因此，要实现 精确的时间和空间控制的src介导的过程调节的AJ，我们建议使用 工程蛋白质工具，将使我们能够精确地调节Src的活性和VE的磷酸化- 活细胞中的钙粘附素。控制级别是史无前例的，因为可以选择性地激活和 在严格的时间控制和活细胞中特定的亚细胞位置失活。重要的是，源 激活可以被限制在特定的下游靶点和亚细胞位置。使用这些工具，我们 将剖析控制AJ组装的单个Src介导的信号通路。

Project Summary Endothelial barrier is regulated at the level of adherens junctions (AJs), cell-cell adhesion structures mediated by the transmembrane protein VE-cadherin. Current model suggests that the tyrosine kinase c-Src functions as a negative regulator of endothelial barrier stimulating disassembly of AJs through phosphorylation of VE cadherin. However, our studies challenge this paradigm. We found that direct activation of Src using our engineered probe transiently enhances barrier function and induces formation of morphologically distinct AJs that exhibit reduced permeability. Our preliminary evidence suggests that Src-induced enhancement of endothelial barrier is mediated through signaling pathway regulating small GTPase Arf6. We also found that phosphorylation of VE cadherin on Tyr658/Tyr731 is critical for barrier strengthening by Src. Our studies also show that Src promotes formation of new contacts with extracellular matrix (focal adhesions) and stimulates interaction of focal adhesion protein p130Cas with VE cadherin. Based on these novel findings we hypothesize that Src signaling though Arf6 and VE-cadherin, and stimulation of new focal adhesions promote formation of AJs leading to strengthening of endothelial barrier. To define the role of each pathway downstream of Src, we propose to address the following questions. 1) We will determine the role of Arf6 activity in Src-stimulated formation of AJs, and identify Src-mediated pathways that activate Arf6. 2) We will define the role of Src signaling in AJs and the role of individual phosphorylation sites on VE-cadherin in formation of new AJs and enhancement of endothelial permeability. 3) We will determine the role of focal adhesions in Src-mediated enhancement of endothelial barrier and define the role of Src signaling through specific focal adhesion proteins. The timing and location of Src-mediated signaling is critical for regulation of AJs. Thus, to achieve precise temporal and spatial control of Src-mediated processes regulating AJs, we propose to employ engineered protein tools that will allow us to regulate precisely the activity of Src and phosphorylation of VE- cadherin in living cells. The level of control is unprecedented in that Src can be selectively activated and inactivated with tight temporal control and in specific subcellular locations in living cells. Importantly, Src activation can be restricted to a specific downstream targets and subcellular locations. Using these tools, we will dissect individual Src-mediated signaling pathways controlling assembly of AJs.