Src-mediated pathways regulating adherens junction assembly.

Src 介导的途径调节粘附连接组装。

• 批准号: 10166863
• 负责人: ANDREI V KARGINOV
• 金额: $ 31.38万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166863
• 关键词: Actins; Address; Adherens Junction; BCAR1 gene; Binding; Blood Vessels; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Complex; Data; Endothelial Cells; Endothelium; Engineering; Event; Exhibits; Extracellular Matrix; Extravasation; Focal Adhesions; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Individual; Integral Membrane Protein; Location; Mediating; Membrane; Modeling; Molecular; Monomeric GTP-Binding Proteins; Morphology; PTK2 gene; Pathologic; Pathway interactions; Permeability; Phosphorylation; Phosphorylation Site; Play; Process; Protein Engineering; Proteins; Regulation; Role; SRC gene; Signal Pathway; Signal Transduction; Site; Structure; Testing; Vascular Endothelium; base; cadherin 5; novel; protein-tyrosine kinase c-src; recruit; src-Family Kinases; tool

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.

项目摘要 内皮屏障在粘附连接（AJs）水平上受到调节，细胞-细胞粘附结构介导了内皮细胞的粘附。 跨膜蛋白VE-钙粘蛋白。目前的模型表明，酪氨酸激酶c-Src的功能是 内皮屏障的负调节剂，通过VE磷酸化刺激AJs分解 钙粘蛋白然而，我们的研究挑战了这种范式。我们发现，直接激活Src使用我们的 工程化探针瞬时增强屏障功能并诱导形成形态学上不同的AJs 其表现出降低的渗透性。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GT3 Arf 6的信号通路介导的。我们还发现 Tyr 658/Tyr 731上的VE钙粘蛋白磷酸化对于Src的屏障强化是关键的。我们的研究还 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 黏着斑蛋白p130 Cas与VE钙粘蛋白相互作用基于这些新发现，我们假设 Src信号通过Arf 6和VE-钙粘蛋白，以及刺激新的局灶性粘连促进形成， AJs导致内皮屏障增强。为了确定Src下游每个途径的作用，我们 提议解决以下问题。1)我们将确定Arf 6活性在Src刺激的细胞中的作用。 形成AJs，并确定Src介导的激活Arf 6的途径。2)我们将定义Src的角色 AJs中的信号传导以及VE-钙粘蛋白上的单个磷酸化位点在新AJs形成中的作用， 内皮通透性增强。3)我们将确定局灶性粘连在Src介导的 增强内皮屏障，并通过特异性粘着斑确定Src信号传导的作用 proteins. Src介导的信号传导的时间和位置对于AJs的调节至关重要。因此，为了实现 精确的时间和空间控制Src介导的过程调节AJs，我们建议采用 工程蛋白工具，使我们能够精确地调节Src的活性和VE的磷酸化， 活细胞中的钙粘蛋白。控制水平是前所未有的，因为Src可以被选择性地激活， 在严格的时间控制下失活，并在活细胞中的特定亚细胞位置失活。重要的是，Src 激活可限于特定的下游靶和亚细胞位置。利用这些工具，我们 将剖析个别Src介导的信号通路控制组装的AJs。